KR20190112100A - Functionalized Poly (propylene Fumarate) Polymer Prepared by Ring Opening Polymerization Using Magnesium Catalyst - Google Patents

Abstract

In one or more embodiments, the present invention relates to a poly (propylene fumarate) functionalized to add a functional group for post-polymerization modification to a bioactive material or other functional species, possibly via one of a number of easy “click” reactions. It relates to polymers and polymer structures (and related methods) made therefrom. In some embodiments, end group functionalization is achieved through the functionalized starting alcohol used in the polymerization process. In some embodiments, functionalization of these poly (propylene fumarate) polymers is achieved through functionalization of propylene oxide monomer precursors, which then form functionalized side chains on the obtained poly (propylene fumarate) polymer.

Description

Functionalized Poly (propylene Fumarate) Polymer Prepared by Ring Opening Polymerization Using Magnesium Catalyst

Cross Reference to Related Application

This application claims the functionality of US Provisional Patent Application Serial No. 62 / 543,786, filed Feb. 2, 2017, entitled “Block Copolymer of Lactone and Poly (propylene Fumarate), filed Feb. 2, 2017. US Pat. Appl. Serial No. 62 / 453,724, filed May 3, 2017, entitled "Copolymerization of Propylene Oxide and Maleic Anhydride Using Mg Catalysts with Initiator," after 3D functionalized polymers for enhanced bioactivity. US Provisional Patent Application Serial No. 62 / 500,777, entitled "Scaffold", US Provisional Patent Application Serial No. 62, filed "functionalized poly (propylene fumarate) polymer and its method of manufacture, filed May 22, 2017) / 509,340, US Provisional Patent Application Serial No. 62 / 541,889, 2017, entitled "Synthesis and Characterization of Well-Defined Poly (propylene Fumarate) and Poly (ethylene Glycol) Block Copolymers, filed Aug. 7, 2017 On September 22, US Patent Application Serial No. 62 / 561,722, entitled "Mg Catalyzed Preparation of Poly (propylene Fumarate) in Hexanes," and "Lactone and Poly (R), filed with the present application on Feb. 2, 2018, Claims the benefit of a US patent application entitled "Block Copolymers of Propylene Fumarate), all of which are incorporated herein by reference in their entirety.

Name of organization for joint research agreement

This application is directed to Akron University, Akron, Ohio and 21 ^st Century Medical Technologies, Inc., Akron, Ohio. Are subject to a joint research agreement.

Field of invention

One or more embodiments of the invention relate to novel poly (propylene fumarate) polymers and methods of making poly (propylene fumarate) polymers. In certain embodiments, the present invention relates to clear biodegradable poly (propylene fumarate) polymers and to scalable methods for making and functionalizing the same. In certain embodiments, the present invention relates to well-defined biodegradable poly (propylene fumarate) polymers for use in various regenerative medicine applications.

As will be appreciated, tissue engineering is an interdisciplinary application of the principles of engineering and life sciences to repair, maintain or improve tissue function, and to develop biological substitutes applicable to the treatment of various therapeutic targets such as blood vessels, nerves and bones. Field. There are two commonly used methods for tissue engineering. One method is to transplant the cells into a support structure device called a scaffold. Another method is to have the scaffold remodeled with natural tissue before the cells transplant it into the patient's body. Based on these tissue engineering principles, scaffolds play an important role in tissue engineering applications. Thus, the materials that make up the scaffold are also important for tissue engineering. There is a need for materials that meet all or almost all requirements for bone tissue engineering and that can be processed to become scaffolds. Common materials for tissue engineering should conform to the bulk mechanical and structural requirements of the target masonry when they form scaffolds and have molecular interactions with cells that promote tissue healing. Furthermore, low toxicity and rapid biodegradation are the basic features that a substance should have.

Materials previously used as scaffolds such as metals, ceramics, natural and synthetic polymers have all failed to meet these requirements. Among these materials, however, synthetic polymers may provide a possible route to suitable scaffold materials due to their ability to adjust their mechanical and physical properties. For example, one type of polyester, poly (propylene fumarate) (PPF), has been synthesized and studied for bone tissue engineering.

In addition, developments in additive manufacturing (such as 3D printing) have the potential to significantly change tissue engineering for a variety of reasons, and the potential to enable these techniques to quickly design and print scaffolds to meet patient specific requirements. It is important to have. However, this development will depend largely on the availability of printable materials to meet the chemical, mechanical and biological requirements of a particular application. Various forms of additive manufacturing known more colloquially as 3D printing have been demonstrated in the literature. Fused Deposition Modeling (FDM) is a multilayer method of extrusion solid filaments such as poly (urethane) (PU), poly ( L -lactic acid) (PLLA) or poly (ester urea) (PEU). In addition, the polymer resin can be printed using continuous digital light source treatment (cDLP), where light-crosslinking in certain areas is achieved through high resolution stereolithography. Inkjet methods have also been demonstrated in 3D printing and can be used with powders or resins.

To produce 3D scaffolds compatible with biological systems, the polymer must be nontoxic, implantable without rejection, and completely reabsorbed upon degradation. The first two criteria are achieved in many polymer systems, while also relatively few examples are bioabsorbable; Polylactide, poly (ε-caprolactone) (PCL) and poly (propylene fumarate) (PPF) are present. Each such example is a polyester and can thus be degraded enzymatically or via in vivo hydrolysis. However, acidosis and inflammation of surrounding tissues are frequently observed as a result of rapid degradation of PLLA. In contrast, slow degradation of PCL in the human body limits its use in tissue healing, particularly related to modifying vascular tissue.

Both PLLA and PCL can be extruded through FDM to produce 3D scaffolds where in vitro degradation can proceed. While the material exhibits moderate mechanical and tensile properties, many defects in the material are observed at the interfaces between the laminated layers. Also, as a result of the attainable width of the extrusion nozzle in FDM, the resolution of the 3D printed scaffold is limited.

However, stereolithographic methods, such as cDLP, exhibit much higher resolution compared to FDM technology because they are limited by the light source of the printer than the materials in which they are used. This allows the 3D scaffold to be printed with controlled porosity that can be adjusted to match physiological conditions.

PPF is an unsaturated polyester that degrades in vivo to form naturally occurring fumaric acid and propylene glycol. PPF has been used for a variety of medical applications such as vascular stents, nerve transplants, cartilage, drug release vehicles, vascular engineering, and bone tissue engineering. As a result of unsaturated alkenes in the polymer backbone, intermolecular crosslinking can be achieved to enhance the mechanical properties of the material. The development of printable PPF resins by dissolving the polymer with the reaction diluent diethyl fumarate (DEF), which acts as both a solvent and a crosslinker, has been extensively studied and it is possible to produce 3D scaffolds with bone-like extrusion modulus. appear.

The current method of PPF production is through phase-growth polycondensation of DEF and propylene glycol (Scheme 1). However, this is not an industrially available production method for PPF for 3D printing purposes. The only low molecular weight PPF (<3000 Da) was demonstrated in the cDLP system as a result of increased crystallinity with chain length leading to solid polymers with low solubility, in contrast to the fluid oligomers observed at lower molecular weights. therefore, Scale-up and commercialization of PPFs for 3D printing through phase-growth polycondensation has proved difficult. Step growth methods do not help to maintain high end group accuracy and narrow molecular weight distribution. Lack of control over the molecular weight distribution directly affects the degradation properties of the material. In addition, residual ethanol and unreacted monomers as by-products of step-grown polycondensation conditions have caused purification problems for industrial scale-up of PPF.

Scheme 1

Polymerization Process for Making Poly (propylene Fumarate)

Ring opening (co) polymerization (ROCOP) of maleic anhydride (MAn) and propylene oxide (PO) has previously been reported to produce PPF cis -alkene isomeric poly (propylene maleate) (PPM), which is a low temperature ( ˜60 ° C.) can be converted to PPF using a weak base. Advantageously, ROCOP allows for a high degree of control over molecular weight distribution and end group accuracy through variations in the ratio of monomer (s) to alcohol initiators. Recent studies of PPF synthesis via ROP, including contact cytotoxicity assays and cell culture results, have shown that PPF polymers produced by ROP are nontoxic and cells have adhered and expanded on their thin films. In the examples in the literature there is already a PPM made using ROCOP, while this system is based on using metal alkoxide catalysts / chain transfer agents with limited options with respect to the alcohol used as the starting end group.

Previously, magnesium ethoxide (Mg (OEt) ₂ ) has been shown to exhibit catalytic behavior for ROCOP formation of PPM. Although not typically used in catalysis as a result of its reactivity, magnesium is attractive for biomaterial synthesis, where terminal oxidation products, magnesium oxide, are used as food additives. However, in the case of Mg (OEt) ₂ , end group accuracy is also not achievable as a result of the weakly coordinated ethoxide ligands acting as chain transfer species. The resulting polymer is an ethoxy- or hydroxy-terminated species. Magnesium 2,6- di -tert-butyl-4-methylphenoxide (Mg (BHT) _2- (THF) ₂ ) is a catalyst previously demonstrated as an 'immortal' ROP catalyst for lactones, which is low Catalytic loading can be used to produce high end group accuracy polyesters. Advantageously, the catalyst shows relative stability in 'air' conditions and does not promote initiation / ester exchange side reactions in the presence of water. Importantly, 2,6- di -tert-butyl-4-methylphenoxide (BHT) was initially selected as a ligand system as a result of its use as antioxidants and stabilizers in food and packaging. Such limited biocompatibility is a concern.

The ability to modulate both functional stoichiometry and degradation properties is important for the utility of PPF. The incorporation of MAn and PO such that PPF is a fully bioabsorbable material can be easily hydrolyzed and avoids side reactions that can incorporate polyether linkers between successive PO repeat units that are not readily degraded. It must be perfectly shifted to produce Controlled degradation is important in many applications where scaffolds can degrade as tissue grows, which maximizes the mechanical contribution of the scaffold and ensures effective replacement with bone tissue. Polymers with a lower molecular weight distribution ( Ð _M ) degrade more uniformly than high Ð _M polymers, which allows better control over scaffold degradation.

In order to minimize transplant rejection by the body and to facilitate the healing process, post-polymerization modification of the surface of the implant with the bioconjugate has been extensively investigated. The addition of small molecules and polypeptides to the scaffold surface has been shown to support cell adhesion and dispersion on the surface of the scaffold. In addition, with the selective selection of polypeptides, the differentiation of these cells can be targeted to produce specified tissue growth ( eg bone tissue). However, scaffolds for tissue growth recovery should affect cell adhesion, proliferation, and differentiation to ensure that only the desired tissue is formed, which has not yet been demonstrated using functionalized PPF. Severe photochemical crosslinking conditions mostly destroy functional groups and limit the ability to incorporate bioactive species using the methods previously described.

However, one of the problems with current PPF polymers is that non-functionalized PPFs do not interact with cells, which is an important requirement for successful biocompatible materials. Therefore, PPF properties have also improved its synthetic method, which already meets some of the basic requirements of bone tissue engineering as a result of its completely bioabsorbable characteristics and lack of observable toxicity, but needs to be further improved. As can be appreciated, a general route for improving synthetic polymer bioactive properties is to functionalize the polymer to obtain or have the desired properties. Since the final goal is to improve PPF bioactivity, one or more functional groups that can undergo a "click" type or other reaction that give the PPF the ability to undergo surface modification and attach helpful bioactive molecules after printing. The ability to add is required.

Thus, what is needed in the art is that a "click" type or other reaction can proceed that has controllable mechanical properties and confers on it surface modification after 3-D printing and confers the ability to attach helpful bioactive molecules to it. Well-defined, non-toxic, biodegradable PPF polymers functionalized with one or more functional groups for PPF as well as methods for the preparation and use of such polymers.

Summary of the Invention

In one or more embodiments, the present invention relates to such PPFs, which may undergo a “click” type or other reaction that proceeds to surface modification after 3-D printing and confers upon it the ability to attach helpful bioactive molecules. Well defined, non-toxic, biodegradable PPF polymers functionalized with one or more functional groups, as well as methods for the preparation and use of such polymers. In some embodiments, these functionalized PPF polymers are formed using a two-step process, wherein the terminal functionalized poly (propylene maleate) (PPM) polymer intermediate has a starting alcohol and magnesium catalyst with functional end groups It is formed in the first step by ring-opening polymerization of maleic anhydride and propylene oxide using. The PPM polymer intermediate is then isomerized in the second step to form a terminal functionalized PPF polymer. In some other embodiments, the present invention provides by functionalizing one of the comonomers used to synthesize PPF via ROP to add functional sites that provide the ability to attach some small bioactive molecules to these PPF materials. A novel functionalized PPF polymer is obtained. Propylene oxide comonomers were chosen for functionalizing these embodiments of the invention, in which alkene bonds from maleic anhydride act as photo-crosslinking sites of the polymer, which is intended to functionalize maleic anhydride comonomers. This makes it difficult to find the reaction site.

In these embodiments, the propylene oxide functionalized comonomers are first obtained using phase transfer chemistry, followed by maleic anhydride and a magnesium catalyst to form new functionalized poly (propylene maleate) polymers after purification. Polymerize with the starting alcohol (which may or may not be functionalized) which is then isomerized to form the novel functionalized PPF polymer of the present invention. The chemical structures of the functionalized comonomers and polymers according to various embodiments of the invention are characterized by ¹ H NMR, ¹³ C NMR, and ¹ H- ¹ H COSY spectroscopy. The high end group accuracy of the polymer was provided by MALDI-ToF mass spectroscopy. In various embodiments of the present invention, the functionalized PPF polymer of the present invention had a molecular weight of about 1000-2000 Da, which is suitable for 3D printing to prepare PPF scaffolds. In addition, these novel functionalized PPF polymers exhibited low dispersion. Furthermore, PPF structures with conventional light crosslinks to produce functionalized PPF scaffolds with strong ability to modify sufficient functional groups attached to these functionalized PPF polymers to complete bone and / or other tissue engineering devices. It has been shown to withstand 3D printing of furnaces.

In a first aspect, the present invention relates to terminal or monomerically functionalized poly (propylene fumarate) polymers. In at least one embodiment, the functionalized poly (propylene fumarate) polymer of the present invention comprises isomerized residues of maleic anhydride monomers and residues of functionalized propylene oxide monomers. In one or more of these embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the invention comprise residues of the functionalized starting alcohol.

In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention having the formula:

Wherein n is an integer from 1 to 1000; R is a functional group selected from the group consisting of alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, tertiary halogen group, and combinations thereof. In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention having the formula:

In formula, n is an integer of 1-1000.

In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention, wherein the functionalized starting alcohol Silver propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropan-2-one, 5-hydroxypentan-2-one, 6-hydroxy Hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, α-bromoisobutyryl 4-methanol benzylmethanoate Or combinations thereof.

In one or more embodiments, the terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention, wherein the functionalized propylene oxide The monomers may be alkyne functionalized propylene oxide, 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO), glycidyl propargyl ether, (±) -epichlorohydrin, or their Selected from combinations. In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention, wherein the functionalized starting alcohol And a functional group selected from alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, tertiary halogen group, and combinations thereof.

In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention having the formula:

Wherein n is an integer from 1 to 100; R is a benzyl group, alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, tertiary halogen group and poly (ethylene glycol) group, or these Is a functional group selected from the combination of; R 'is a functional group or an alkyl or aryl group having a functional group, said functional group being an alkyne group, alkene group, hydroxyl group, protected hydroxyl group, thiol group, halide group, or a combination thereof. In one or more embodiments, terminal or monomeric functionalized poly (propylene fumarate) polymers of the present invention comprise any one or more of the aforementioned embodiments of the first aspect of the present invention having the formula selected from:

In formula, n is an integer of 1-100.

In at least one embodiment, the terminal or monomeric functionalized poly (propylene fumarate) polymer of the present invention has a number average molecular weight ( M _n ) of about 0.7 kDa to about 100,000 kDa as mentioned above in the first aspect of the present invention. Any one or more of the embodiments. In at least one embodiment, the terminal or monomeric functionalized poly (propylene fumarate) polymer of the present invention has the above-mentioned embodiment of the first aspect of the invention having a polydispersity index ( Ð _M ) of about 1.01 to about 1.8. Any one or more of the examples.

In a second aspect, the present invention provides a functionalization for forming a monomer functionalized poly (propylene fumarate) polymer comprising a glycidyl group linked directly or via an ether bond to a functional group or an alkyl or aryl group comprising the functional group. Propylene oxide monomer, wherein a functional group can be introduced into the click reaction with the corresponding functional group. In one or more of these embodiments, the functional group is selected from an alkyne group, alkene group, hydroxyl group, protected hydroxyl group, or a combination thereof.

In one or more embodiments, the functionalized monomers of the present invention comprise any one or more of the aforementioned embodiments of the second aspect of the present invention, wherein the functionalized monomers are glycidyl propargyl ethers. In one or more embodiments, the functionalized monomers of the invention comprise any one or more of the aforementioned embodiments of the second aspect of the invention having the formula:

In one or more embodiments, the functionalized monomers of the present invention comprise any one or more of the above-mentioned embodiments of the second aspect of the present invention, wherein the functionalized monomers are 2-[[(2-nitrophenyl ) Methoxy] methyl] oxirane (NMMO). In one or more embodiments, the functionalized monomers of the invention comprise any one or more of the aforementioned embodiments of the second aspect of the invention having the formula:

In a third aspect, the invention relates to a functionalized propylene oxide monomer for forming the monomer functionalized poly (propylene fumarate) polymer of the first aspect of the invention having the formula:

Wherein R is a functional group or an alkyl or aryl group comprising a functional group, which is selected from an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, a thiol group, a halide group, or a combination thereof. In one or more of these embodiments, the functionalized propylene oxide monomer has the formula:

In a fourth aspect, the invention relates to a functionalized propylene oxide monomer for forming the monomer functionalized poly (propylene fumarate) polymer of the first aspect of the invention having the formula:

In a fifth aspect, the present invention relates to a method for preparing the terminal or monomeric functionalized poly (propylene fumarate) polymer of the first aspect of the invention, comprising: a disclosure further comprising a functional end group Preparing alcohol; Combining the starting alcohol, magnesium catalyst, maleic anhydride, and propylene oxide in a suitable vessel and adding a suitable solvent; The vessel is sealed and then heated to cause and / or maintain a ring-opening polymerization reaction between the maleic anhydride and propylene oxide initiated by the starting alcohol, thereby producing a poly (propylene maleate) polymer comprising functional end groups. Forming; Collecting and purifying the poly (propylene maleate) polymer comprising functional end groups; And isomerizing the poly (propylene maleate) polymer comprising the functional end group to the poly (propylene fumarate) polymer comprising the functional end group. In one or more of these embodiments, the molar ratio of starting alcohol to magnesium catalyst in the combination is about 1: 1.

In one or more embodiments, the process of the invention comprises any one or more of the abovementioned embodiments of the fifth aspect of the invention, wherein the monomers (maleic anhydride and functionalized and / or unfunctionalized propylene oxide The ratio of moles of starting alcohol to the total moles of) is from about 1: 5 to about 1: 1000. In at least one embodiment, the process of the invention comprises any one or more of the abovementioned embodiments of the fifth aspect of the invention, wherein the total monomer concentration (maleic anhydride and functionalized and / or Unfunctionalized propylene oxide) is about 0.5M to about 5.0M. In at least one embodiment, the process of the invention comprises any one or more of the abovementioned embodiments of the fifth aspect of the invention, wherein a suitable solvent is toluene or hexane.

In one or more embodiments, the method of the present invention comprises any one or more of the aforementioned embodiments of the fifth aspect of the present invention, wherein the heating step heats the vessel to a temperature of about 40 ° C to about 100 ° C. It involves doing. In at least one embodiment, the process of the invention comprises any one or more of the abovementioned embodiments of the fifth aspect of the invention, wherein the suitable solvent is hexane and the heating step is carried out at a temperature of about 45 ° C. Heating.

In at least one embodiment, the process of the invention comprises any one or more of the abovementioned embodiments of the fifth aspect of the invention, wherein the starting alcohol is benzyl alcohol, propargyl alcohol, allyl alcohol, 4-di Benzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropane-2-one, 5-hydroxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptane 2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, α-bromoisobutyryl 4-methanol benzylmethanoate, and combinations thereof. In one or more embodiments, the methods of the present invention comprise any one or more of the aforementioned embodiments of the fifth aspect of the invention, wherein the functional end groups of the starting alcohol are alkyne groups, propargyl groups, allyl groups , Alkene groups, 4-dibenzocyclooctyne groups, cyclooctyne groups, ketone groups, aldehyde groups, tertiary halogen groups, poly (ethylene glycol) groups, and combinations thereof. In one or more embodiments, the process of the invention comprises any one or more of the above-mentioned embodiments of the fifth aspect of the invention, wherein the magnesium catalyst comprises Mg (BHT) ₂ (THF) ₂ .

In a sixth aspect, the present invention relates to a method for preparing the functionalized poly (propylene fumarate) polymer of the first aspect of the present invention comprising: preparing a functionalized propylene oxide; Reacting propylene oxide functionalized in the presence of a magnesium catalyst with maleic anhydride and starting alcohol to form a functionalized poly (propylene maleate) polymer; Isomerizing the functionalized poly (propylene maleate) polymer by reacting it with a base to form the functionalized poly (propylene fumarate) polymer of the first aspect of the present invention. In one or more of these embodiments, the functionalized propylene oxide is selected from alkyne functionalized propylene oxide, 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO), glycidyl propargyl ether, ( ±) -epichlorohydrin or a combination thereof. In some of these embodiments, the starting alcohol is benzyl alcohol, propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropan-2-one, 5-hydroxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, α -Bromoisobutyryl 4-methanol benzylmethanoate, and combinations thereof. In one or more of these embodiments, the metal catalyst is magnesium 2,6- di -tert-butyl-4-methylphenoxide (Mg (BHT) ₂ (THF) ₂ ).

In a seventh aspect, the present invention relates to a method of preparing the functionalized monomer of the first aspect of the present invention comprising: sodium hydroxide (NaOH), potassium hydroxide (KOH), and combinations thereof Adding propargyl alcohol to an aqueous solution containing a base selected from; Dissolving (±) -epichlorohydrin and tetrabutylammonium hydrogensulfate in a suitable organic solvent; Adding a solution and H ₂ O to the propargyl alcohol solution; And proceeding the reaction under an inert atmosphere to prepare glycidyl propargyl ether.

In some of these embodiments, adding propargyl alcohol comprises adding dropwise propargyl alcohol to an aqueous solution of sodium hydroxide (NaOH) at a temperature of about −10 ° C. to about 30 ° C. while stirring. The aqueous solution of sodium hydroxide (NaOH) comprises from about 20% to about 50% by weight NaOH; The organic solvent in the dissolution step comprises hexane; Advancing the reaction includes increasing the reaction temperature to ambient temperature and continuing the reaction for about 1 hour to about 24 hours under an N ₂ blanket. In some other embodiments, the method further comprises: quenching the reaction; Extracting the crude product with a suitable organic solvent; And purifying the crude product by column chromatography or distillation to produce purified glycidyl propargyl ether.

In an eighth aspect, the present invention relates to a method of preparing a functionalized monomer comprising: dissolving o -nitrobenzyl alcohol in a suitable organic solvent; Adding an aqueous solution containing tetrabutylammonium hydrogensulfate and a base to an o -nitrobenzyl alcohol solution, the base selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), and combinations thereof step; Adding (±) -epichlorohydrin; And advancing the reaction to produce 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO). In one or more of these embodiments, the method further comprises the following steps: extracting the crude product with a suitable organic solvent; And purifying the crude product by column chromatography or distillation to produce purified 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO).

In another aspect, the invention relates to a 3-D printed polymer scaffold comprising the functionalized poly (propylene fumarate) polymer of the first aspect of the invention. In one or more of these embodiments, the 3-D printed polymer scaffold further comprises a plurality of bioactive materials bound to the functionalized poly (propylene fumarate) polymer.

(중공 형상)을 나타내는 그래프이다. 분자량은

표준에 대한 SEC에 의해 결정된다.
도 7은 DP 10 벤질 알코올 개시된 PPM (표 2, 항목 1)에 대한 ¹³C NMR 스펙트럼이다 (125 MHz, CDCl₃, 303 　K).
도 8은 DP 25 벤질 알코올 개시된 PPM (표 2, 항목 2)에 대한 SEC 크로마토그램이다. 분자량은 폴리(스티렌) 표준에 대해 결정된다.
도 9는 DP 25 프로파르길 알코올 개시된 PPM (표 2, 항목 6)에 대한 ¹³C NMR 스펙트럼이다 (125 MHz, DMSO-d ₃, 303 　K).
도 10은 DP 25 프로파르길 알코올 개시된 PPM (표 2, 항목 6)에 대한 SEC 크로마토그램이다. 분자량은 폴리(스티렌) 표준에 대해 결정된다.
도 11은 DP 25 4-하이드록시부탄-2-온 개시된 PPM (표 2, 항목 10)에 대한 ¹³C NMR 스펙트럼이다 (125 MHz, CDCl₃, 303 　K).
도 12는 DP 25 4-하이드록시부탄-2-온 개시된 PPM (표 2, 항목 10)에 대한 SEC 크로마토그램이다. 분자량은 폴리(스티렌) 표준에 대해 결정된다.
도 13은 60℃에서 헥산 중에 중합된 DP 25 벤질 알코올 개시된 PPM (표 3, 항목 2)의 ¹H NMR 스펙트럼이다 (300 MHz, CDCl₃, 303 　K).
도 14는 60℃에서 헥산 중에 중합된 DP 25 벤질 알코올 개시된 PPM (표 3, 항목 2)의 ¹³C NMR 스펙트럼이다 (125 MHz, CDCl₃, 303 　K).
도 15는 45℃에서 헥산 중에 중합된 DP 25 벤질 알코올 개시된 PPM (표 3, 항목 2)에 대한 SEC 크로마토그램이다. 분자량은 폴리(스티렌) 표준에 대해 결정된다.
도 16은 아자이드-작용화된 GRGDS (N₃-GRGDS) 폴리펩타이드 서열에 대한 ESI 스펙트럼이다.
도 17은 라이브/데드® 검정에 의해 결정되는 프로파르길 알코올-작용화된 PPF 및 GRGDS-PPF 바이오콘주게이트 필름에 대한 프로파르길 알코올-작용화된 PPF, 물리적으로 흡착된 N₃-GRDGS의 세포 생존비를 나타내는 그래프이다.
도 18은 글리시딜 프로파르길 에테르 (하부), 프로파르길 알코올 (중간) 및 에피클로로히드린 (상부)의 ¹H NMR 스펙트럼이다.
도 19는 2-[[(2-니트로페닐)메톡시]메틸]옥시란의 ¹H NMR 스펙트럼이다.
도 20은 트랜스-폴리(글리시딜 프로파르길 에테르-코-말레산 무수물)의 ¹H NMR 스펙트럼이다.
도 21은 트랜스-폴리(글리시딜 프로파르길 에테르-코-말레산 무수물)의 정량적 ¹³C NMR 스펙트럼이다.
도 22는 이성질체화 이전 (하부) 및 이후 (상부)의 폴리(2-[[(2-니트로페닐)메톡시]메틸]옥시란-코-말레산 무수물)의 ¹H NMR 스펙트럼이다.
도 23은 트랜스-폴리(2-[[(2-니트로페닐)메톡시]메틸]옥시란-코-말레산 무수물)의 ¹H NMR 스펙트럼이다.
도 24는 트랜스-폴리(2-[[(2-니트로페닐)메톡시]메틸]옥시란-코-말레산 무수물)의 ¹³C NMR 스펙트럼이다.
도 25는 글리시딜 프로파르길 에테르 (GPE) 및 말레산 무수물 (MA)의 공중합의 단기 동력학 연구 플롯이다.
도 26은 글리시딜 프로파르길 에테르 (GPE) 및 말레산 무수물 (MA)의 공중합의 장기 동력학 연구 플롯이다.
도 27은 2-[[(2-니트로페닐)메톡시]메틸]옥시란 (NMMO) 및 말레산 무수물 (MA)의 공중합의 동력학 연구 플롯이다.
도 28은 폴리(ECH-코-MA)의 ¹H NMR 스펙트럼이다 (300 MHz, 303 　K, CDCl₃).
도 29는 폴리(ECH-코-MA)의 정량적 ¹³C NMR 스펙트럼이다 (125 MHz, 303 　K, CDCl₃).
도 30은 P(ECH-코-MA)의 분자량 분포의 SEC 크로마토그램이다.
도 31은 폴리(GPE-코-MA)의 ¹H NMR 스펙트럼이다 (300 MHz, 303 　K, CDCl₃).
도 32는 폴리(GPE-코-MA)의 정량적 ¹³C NMR 스펙트럼이다 (125 MHz, 303 　K, CDCl₃).
도 33은 폴리(NMMO-코-MA)의 ¹H NMR 스펙트럼이다 (300 MHz, 303 　K, CDCl₃).
도 34는 폴리(NMMO-코-MA)의 ¹³C NMR 스펙트럼이다 (125 MHz, 303 　K, CDCl₃).DETAILED DESCRIPTION For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention in conjunction with the following accompanying drawings:
1 is a ¹ H NMR spectrum of DP 10 benzyl alcohol initiated poly (propylene maleate) (Table 2, item 1) using Mg (BHT) ₂ (THF) ₂ as catalyst (300 MHz, 303 K, DMSO- d ₆ ).
FIG. 2 is a ¹ H NMR spectrum of DP 25 propargyl alcohol initiated poly (propylene maleate) (Table 2, item 6) using Mg (BHT) ₂ (THF) ₂ as catalyst (300 MHz, 303 K, DMSO- d ₆ ).
FIG. 3 is a ¹ H NMR spectrum of DP 25 4-hydroxybutan-2-one disclosed poly (propylene maleate) (Table 2, item 10) using Mg (BHT) ₂ (THF) ₂ as catalyst (300 _{MHz, 303 K, DMSO- d 6} ).
FIG. 4 is a ¹ H NMR spectrum comparison of (top) precursor DP25 propargyl alcohol-initiated PPM produced in hexane (Table 2, item 6) and propargyl alcohol-initiated PPF obtained (bottom) after isomerization ( 500 MHz, CDCl ₃ , 303 K).
5a-b shows maleic acid carried out at 80 ° C. in toluene with [MAn] ₀ : [PO] ₀ : [BnOH] ₀ : [catalyst] ₀ = 25: 25: 1: 1, total initial monomer concentration = 2 M Kinetics plots for copolymers of anhydride and propylene oxide (FIG. 5A) and M _n (diamond form) and Ð _M (square) for increased monomer conversion for the same copolymer as determined by SEC for poly (styrene) standards A graph showing the change of (FIG. 5B).
6 shows M _n (solid shape) for PPF at varying degrees of polymerization using a range of disclosed species;

It is a graph showing (hollow shape). Molecular weight is

Determined by the SEC for standards.
FIG. 7 is a ¹³ C NMR spectrum for DP 10 benzyl alcohol initiated PPM (Table 2, item 1) (125 MHz, CDCl ₃ , 303 K).
8 is SEC chromatogram for DP 25 benzyl alcohol initiated PPM (Table 2, item 2). Molecular weights are determined against poly (styrene) standards.
FIG. 9 is a ¹³ C NMR spectrum for DP 25 propargyl alcohol initiated PPM (Table 2, item 6) (125 MHz, DMSO- d ₃ , 303 K).
10 is SEC chromatogram for DP 25 propargyl alcohol initiated PPM (Table 2, item 6). Molecular weights are determined against poly (styrene) standards.
FIG. 11 is ¹³ C NMR spectrum for DP 25 4-hydroxybutan-2-one initiated PPM (Table 2, item 10) (125 MHz, CDCl ₃ , 303 K).
12 is a SEC chromatogram for DP 25 4-hydroxybutan-2-one initiated PPM (Table 2, item 10). Molecular weights are determined against poly (styrene) standards.
FIG. 13 is a ¹ H NMR spectrum of DP 25 benzyl alcohol initiated PPM (Table 3, item 2) polymerized in hexane at 60 ° C. (300 MHz, CDCl ₃ , 303 K).
FIG. 14 is a ¹³ C NMR spectrum of DP 25 benzyl alcohol initiated PPM (Table 3, item 2) polymerized in hexane at 60 ° C. (125 MHz, CDCl ₃ , 303 K).
FIG. 15 is a SEC chromatogram for DP 25 benzyl alcohol initiated PPM (Table 3, item 2) polymerized in hexane at 45 ° C. FIG. Molecular weights are determined against poly (styrene) standards.
Figure 16 is an ESI spectrum for an azide-functionalized GRGDS (N ₃ -GRGDS) polypeptide sequence.
FIG. 17 shows propargyl alcohol-functionalized PPF, physically adsorbed N ₃ -GRDGS for propargyl alcohol-functionalized PPF and GRGDS-PPF bioconjugate films as determined by Live / Dead® Assay. It is a graph showing the cell survival ratio.
Figure 18 is a ¹ H NMR spectrum of glycidyl propargyl ether (bottom), propargyl alcohol (middle) and epichlorohydrin (top).
19 is a ¹ H NMR spectrum of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane.
20 is a ¹ H NMR spectrum of trans- poly (glycidyl propargyl ether- co -maleic anhydride).
21 is a quantitative ¹³ C NMR spectrum of trans- poly (glycidyl propargyl ether- co -maleic anhydride).
FIG. 22 is a ¹ H NMR spectrum of poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride) before (bottom) and after (top) isomerization.
Figure 23 is a ¹ H NMR spectrum of trans- poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride).
Figure 24 is a ¹³ C NMR spectrum of trans- poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride).
25 is a short-term kinetic study plot of the copolymerization of glycidyl propargyl ether (GPE) and maleic anhydride (MA).
FIG. 26 is a long-term kinetic study plot of the copolymerization of glycidyl propargyl ether (GPE) and maleic anhydride (MA).
FIG. 27 is a kinetics study plot of the copolymerization of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO) and maleic anhydride (MA).
FIG. 28 is a ¹ H NMR spectrum of Poly (ECH-CO - MA) (300 MHz, 303 K, CDCl ₃ ).
29 is a quantitative ¹³ C NMR spectrum of Poly (ECH-CO - MA) (125 MHz, 303 K, CDCl ₃ ).
Fig. 30 is a SEC chromatogram of the molecular weight distribution of P (ECH- co- MA).
31 is a ¹ H NMR spectrum of poly (GPE- nose -MA) (300 MHz, 303 K , CDCl 3).
Figure 32 is a quantitative ¹³ C NMR spectrum of the poly (GPE- nose -MA) (125 MHz, 303 K , CDCl 3).
33 is a ¹ H NMR spectrum of poly (NMMO- nose -MA) (300 MHz, 303 K , CDCl 3).
Figure 34 is a ¹³ C NMR spectrum of the poly (NMMO- nose -MA) (125 MHz, 303 K , CDCl 3).

As set out above, due to its low cytotoxicity, good biodegradability and controllable mechanical properties, poly (propylene fumarate) (PPF) is an ideal synthetic polyester for preparing scaffolds for bone or other tissue engineering. However, based on tissue engineering requirements, PPFs should ideally have molecular interactions with cells to support cell adhesion, proliferation and differentiation. Therefore, the functionalization of PPF to attach bioactive molecules ( ie bioactive drugs, peptides, proteins, sugars) is an important step when used for bone tissue engineering applications. One of the easier ways to modify synthetic polymers is to add functional groups for post-polymerization modification, which will alter the chemical structure of the polymer. Based on the PPF chemical structure and its synthetic route ( ie ROCOP), there are two potential methods for functionalization. One is end group functionalization via the functionalized starting alcohol, and the other is through the functionalization of the monomer precursor, which then forms functionalized side chains on the PPM / PPF polymer upon polymerization ("monomer functionalized "PPM / PPF).

As used herein, the term “functionalized” refers to a polymer, bioactive material, or other material that includes or is modified to include a functional group, and the broader term “functionalized” refers to Processes, methods and / or reactions added to polymers, bioactive materials, or other materials, and in particular the addition of functional groups to the PPF polymer and / or to the PPF polymer, even when added before isomerization Or a bioactive material or other functional species for the purpose of adding another functional species. Also, as used herein, the terms “functional group” and “functional moiety” are used interchangeably to refer to chemically active species or groups containing chemically active species. As used herein, a "functionalized poly (propylene fumarate) polymer" is a poly (propylene fumarate) comprising one or more functional groups for the purpose of adding bioactive materials of other functional species to the polymer. Refers to a polymer.

Thus, the term “terminal functionalized,” or “terminal functionalized” or “terminal functionalized,” are used interchangeably to refer to polymers modified to have or contain functional groups at the ends of the polymer chains. The term “terminal functionalization,” or “terminal functionalization” or “terminal functionalization,” are used interchangeably to refer to processes, methods and / or reactions in which functional groups are added to the ends of the polymer chain. Used. As used herein, the term “monomer functionalized” refers to a PPM or PPF polymer having functionalized monomers, and in particular one or more functional groups added in the course of polymerization via the functionalized propylene oxide monomer. Similarly, as used herein, the term "functional end group" refers to a functional group located at the end of a polymer chain. The term “functionalized initiating alcohol” refers herein to an alcohol capable of initiating ring-opening copolymerization of maleic anhydride monomers and functionalized or unfunctionalized polypropylene oxide monomers in the presence of a magnesium catalyst.

As used herein, the terms "bioactive molecule (s)" and "bioactive material (s)" refer to substances that affect cellular function. Bioactive molecules can include, but are not limited to, peptides, carbohydrates, proteins, oligonucleotides, and small molecule drugs.

The term isomerization refers herein to the conversion of cis -isomer (PPM) to trans- isomer (PPF) form in the presence of a catalyst.

The bioactive or other functional species that can be attached to the functional groups on the PPF polymer of the present invention are not particularly limited, provided that they contain or are functionalized to contain a moiety that can be bound to at least one of the functional groups on the polymer. . As used herein, the terms “bioactive molecule (s)” and “bioactive material (s)” refer to substances that affect cellular function, including but not limited to peptides, carbohydrates, proteins, oligonucleotides, and Small molecule drugs. As used in the context of the use of materials that can be attached to the PPF polymer of the present invention, the term "functional species" can be added to the functionalized PPF polymer of the present invention to provide added benefits. It refers to materials other than and may include such as fluorescent and other markers, small molecule dyes, and / or halide atoms. The bioactive or other functional species to be attached are not limited in size, while they are generally less than about 40,000 Da, and they cannot be easily reached on the inner surface of the printed or formed polymer structure and / or enable bonding In such a way that the functional groups on the PPF polymer should not be so large that they cannot be reached. In various embodiments, the bioactive or other functional species to be attached are, but are not limited to, short chain peptides, peptides, proteins, sugars, carbohydrates, bioactive drugs, oligonucleotides, small molecule drugs, fluorescent or other markers, small molecule dyes and / or And halide atoms.

Although not necessary to practice the present invention, the functional groups on the PPF polymer of the present invention are preferably functional groups capable of entering a well-known "click" reaction to facilitate post-polymerization addition of the desired materials, such as bioactive compounds, to the polymer. to be. As used herein, the terms "click reaction," "click chemistry," "click chemistry method," and "click chemistry reaction," are generally referred to in the art as "click" reactions that meet the following prerequisites: Used interchangeably to refer to a group of orthogonal conjugation reactions: (i) high yield, near quantitative conversion; (ii) biologically good conditions (aqueous solution, ambient temperature, and near physiological pH); (iii) Residual by-products, with or without limitation. These reactions are typically performed simply in a wide range, in high yield, stereospecific, and produce only byproducts that can be removed without chromatography, and can be carried out in solvents that are easily removable or mild. Similarly, the term "clickable" refers to a molecule or functional group capable of binding via a click reaction.

As suggested above, the concept of "click" chemistry currently represents a number of orthogonal reactions, which are powerful, selective, efficient, and high in yield. In various embodiments, suitable click reactions include, but are not limited to, copper (I) copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) reactions (also known as Huisgen cyclization reactions), thiol- En radical addition reaction, oxime ligation reaction, Michael-addition reaction, thiol-michael-addition reaction, Mannich-type addition reaction, "ene-type" addition reaction, thiol-ene radical addition, modification-promoted azide-alkyne Cycloaddition (SPAAC) Reaction, Non-Stickless Studdinger Ligation, Spotless Studdinger Ligation, Diels-Alder Reaction, Hetero Diels-Alder Reaction, Reverse Electron Demand Diels-Alder Reaction, Tandem [3 + 2 Cycloaddition-retro-dils-alder (tandem crD-A) reaction, thiol-alkyne reaction, thiol-pyridyl disulfide reaction, thiol-halogen ligation, native chemical ligation, and thiozolidine And a ligation reaction. In one or more embodiments, suitable "clickable" moieties may include, but are not limited to, alkyne, alkene, azide, ketone or modified cyclooctyne groups. In at least one embodiment, the bioactive or other functional species is a thiol-ene reaction, a thiol-phosphorus reaction, a 1,3-dipolar cycloaddition between alkyne and azide, or action on a bioactive or other functional species. It can be attached to the functional groups on the PPF polymer of the invention by oxime ligation between the ketone and amine type reactions between the sex moiety and the functional groups on the functionalized PPF polymer of the invention.

End-functionalized PPF Polymer

In a first aspect, the present invention is non-toxic and has a limited and expected material property suitable for 3D printing using a reaction initiator and is terminally functionalized which can be induced into a bioactive group after it has been printed or formed a polymer structure. It relates to a poly (propylene fumarate) polymer. As set forth above, in some embodiments the PPF polymer of the invention contains functional end groups useful for the post-polymerization reaction to add functional species to the polymer, which forms the PPM intermediate in the first step described above. Is introduced into these PPF polymers via an initiating alcohol which is used to. The terms "initiating alcohol" and "initiating alcohol" refer to a molecule comprising a hydroxyl group which initiates a ring-opening polymerization reaction of maleic anhydride and propylene oxide in the presence of an Mg catalyst, which is directly or indirectly bound to a functional end group. Used interchangeably to refer to. Some or all of these functional end groups withstand both polymerization (step 1) and isomerization reactions (step 2) to form the PPF polymer of the present invention, and at least one functional species such as bioactive materials, markers, small molecule dyes Useful for post-polymerization addition of short chain peptides, drugs, and / or halide atoms. As used herein, the term “isomerization” broadly refers to the conversion of cis -isomer (PPM) to its trans- isomer (PPF) form, or chemical reaction or process (“isomerization reaction”). ") Refers to a reaction or process that converts the cis -isomer (PPM) to its trans- isomer (PPF) form.

The functional end groups which can be used are not particularly limited, provided that at least part of their reactivity is maintained after the polymerization and isomerization reaction. In at least one embodiment, the functional groups on the terminal functionalized PPF polymer of the present invention include, but are not limited to, benzyl groups, alkyne groups, propargyl groups, allyl groups, alkenes groups, 4-dibenzocyclooctin groups, cyclooctyne groups, Ketone groups, aldehyde groups, tertiary halogen groups and poly (ethylene glycol) groups, lactone groups, protected hydroxyl groups, or combinations thereof. As used herein, the term “protected hydroxyl group” refers to a hydrogen atom as a protecting group to prevent unwanted reactions until the desired reaction occurs, at which point the protecting group is replaced with a proton to re-form the hydroxyl group. Refers to the hydroxyl group being replaced. Any suitable protecting group or protecting groups known in the art can be used, including but not limited to tert -butyloxycarbonyl (BOC) groups, trimethylsilyl ether (TMS) groups, tert -butyldimethylsilyl ether (TBDMS), or Fluorenylmethyloxycarbonyl (FMOC) groups.

In one or more embodiments, the PPF polymers of the present invention may have more than one different terminal functional group. In some embodiments, this can be accomplished using starting alcohols having more than one type of functional group. However, this method will result in polymer mixtures with broader polydispersity and lower control over polymer properties than when a single functionalized starting alcohol is used. Thus, the preparation of the PPF polymers of the invention having more than one different terminal functional groups preferably produces a batch of two or more separate polymers each having different desired terminal functional groups, which are then combined into a homogeneous mixture to 1 It is achieved by forming a PPF polymer of the invention which may have more than two different terminal functional groups.

In various embodiments, terminally functionalized PPF polymers of the invention have a number average molecular weight ( M _n ) of about 0.7 kDa to about 100,000 kDa and a polydispersity of about 1.01 to about 1.8 as measured by size exclusion chromatography (SEC). Will also have an exponent ( Ð _M ). In some embodiments, the terminal functionalized PPF polymer is at least 1.0 kDa, in other embodiments, at least about 5 kDa, in other embodiments, at least about 8 kDa, in other embodiments, at least about 10 kDa, in other embodiments , At least about 1000 kDa and in other embodiments, at least about 10,000 kDa M _n . In some embodiments, the terminal functionalized PPF polymer is 90,000 kDa or less, in other embodiments, about 80,000 kDa or less, in other embodiments, about 70,000 kDa or less, in other embodiments, about 60,000 kDa or less, in other embodiments , Up to about 50,000 kDa, in other embodiments, up to about 40,000 kDa M _n .

In some embodiments, the terminal functionalized PPF polymer has at least 1.03, in other embodiments, at least about 1.05, in other embodiments, at least about 1.10, in other embodiments, at least about 1.20, in other embodiments, at least about 1.30. , In other embodiments, it will have a polydispersity index ( Ð _M ) of about 1.40 or greater. In some embodiments, the terminal functionalized PPF polymer is at most 1.70, in other embodiments, at most about 1.60, in other embodiments, at most about 1.50, in other embodiments, at most about 1.40, in other embodiments, at most about 1.30 , And in other embodiments, will have a Ð _M of about 1.20 or less.

In various embodiments, terminally functionalized poly (propylene fumarate) polymers of the present invention may have the following formula:

Wherein n is an integer from 1 to 1000 and R is a useful functional group for the post-polymerization addition of a preferred substance, such as a bioactive compound, to the polymer. In some of these embodiments, n is from 100 to 1000, in another embodiment, from 200 to 1000, in other embodiments, from 300 to 1000, in other embodiments, from 400 to 1000, in other embodiments, from 1 to 900, In other embodiments, it is an integer from 1 to 800, in other embodiments, from 1 to 700, and in other embodiments, from 1 to 600. In various embodiments, R is, but is not limited to, a benzyl group, alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, tertiary halogen group and Poly (ethylene glycol) groups, and combinations thereof. In some of these embodiments, R may comprise a clickable moiety as defined above.

In some embodiments, the poly (propylene fumarate) polymer of the present invention may have the following formula:

Wherein n is defined above.

As set forth above, the terminal functional groups of the PPF of the present invention can be used for the post-polymerization addition of functional species, such as bioactive or other useful materials, to the polymer. In one or more of these embodiments, the functional species attached to the PPF polymer of the invention contain functional groups that can be bound to terminal functional groups of the PPF by click or other type of reaction without naturally losing their desired functionality. something to do. However, in some other embodiments, the functional species attached to the PPF polymer of the present invention must first be functionalized with a moiety known to bind to terminal functional groups, preferably via a click reaction. The particular clickable moiety and means for its attachment to the functional species will depend on the bioactive or other substance to which it is attached and the specific click reaction used. One skilled in the art will be able to attach the appropriate clickable moiety to the bioactive or other material to which it is attached without undue experimentation. For example, a peptide functionalized with an azide group can be coupled to an alkyne end-functionalized PPF chain on a scaffold using a Huisgen 1,3 cycloaddition reaction. For example, peptides functionalized with thiol groups or cysteine residues can be coupled to alkenes or norbornene end-functionalized PPF chains on scaffolds using thiolene reactions.

The addition of terminal functional groups on the PPF polymer of the present invention has been found to have no significant effect on the desired mechanical, thermal, degradation, and / or toxic properties of the polymer at the molecular weights disclosed and discussed herein.

Monomer-functionalized PPF polymer

In a second aspect, the present invention relates to a novel functionalized PPF polymer comprising isomerized residues of maleic anhydride monomers and residues of functionalized propylene oxide monomers used to form the polymer. As is apparent, when the residues of the maleic anhydride monomer and the functionalized propylene oxide monomer react to form a polymer, the maleic anhydride monomer and propylene oxide have the functional groups of the functionalized propylene oxide monomer forming an active side chain. Will form the backbone of the PPM / PPF polymer.

As used herein, the term “residue (s)” is generally used to refer to a portion of a monomer or other chemical unit that is incorporated into a polymer or macromolecule. Further, the terms "residue of maleic anhydride monomer" and "residue of functionalized propylene oxide monomer" are used to refer to the portion of maleic anhydride monomer and functionalized propylene oxide monomer incorporated into the PPM and PPF polymer, respectively. . The term “isomerized moiety of maleic anhydride monomer” specifically refers to the residue of the maleic anhydride monomer in which the double bond isomerized from the cis configuration to the trans configuration to form a PPF polymer. The term “residue of starting alcohol” or “initiating alcohol residue” and the like refers to the portion of the starting alcohol that remains bound at the end of the PPM / PPF polymer chain after it commences the polymerization. Similarly, the term “residue of functionalized initiating alcohol” or “functionalized initiating alcohol residue” or the like refers to a functionalized initiating alcohol in which the functionalized initiating alcohol remains bound at the end of the polymer chain after initiation of the polymerization. It refers to a functional group or other part of.

In various embodiments, the functional groups on the residues of the functionalized propylene oxide monomer include, but are not limited to, alkyne groups, propargyl groups, alkenes groups, hydroxyl groups, ketone groups, thiol groups, halide groups, nitrobenzyl groups, or functional groups such as Halide groups, nitrobenzyl groups, or groups that can be easily converted to hydroxyl groups. The use of monomer functionalization methods has been found to increase the amount of functional groups available compared to unique end group functionalization even using less functional groups that withstand the process. In addition, it has been found that the monomers of the PPF polymers of the present invention have no significant effect on the desired mechanical, thermal, deterioration, and / or toxic properties of the polymers at the molecular weights disclosed and discussed herein.

In various embodiments, the functionalized propylene oxide monomer residues of the novel functionalized PPF polymers of the invention may be introduced into a "click" reaction with a bioactive compound functionalized with or with a functional group corresponding to the click reaction. It will contain functional groups that can be used. For example, in one or more of these embodiments, the functional group on the functionalized propylene oxide can be an alkyne group, alkene group, hydroxyl group, ketone group, thiol group, or a group that can be readily converted to such functional group. In some embodiments, the functionalized propylene oxide can be (±) -epichlorohydrin. In one or more embodiments, the functionalized propylene oxide can be an alkyne functionalized propylene oxide. In some embodiments, the novel functionalized PPF polymers of the invention comprise residues of glycidyl propargyl ether.

In some of these embodiments, the novel functionalized PPF polymers of the invention comprise residues of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO). In these embodiments, it is to be understood that the nitrobenzyl group on the NMMO is a UV sensitive protecting group, which can be easily replaced by hydroxyl groups upon exposure to certain UV wavelengths. In some other embodiments, the novel functionalized PPF polymers of the invention comprise residues of halide functionalized propylene oxide. In some of these embodiments, the novel functionalized PPF polymers of the invention comprise residues of (±) -epichlorohydrin. In these embodiments, it is to be understood that the halide group on the (±) -epichlorohydrin is a protecting group, which is then replaced by any suitable nucleophile. Suitable nucleophiles can include, but are not limited to, amines, alcohols, thiols and hydroxylamines.

As is also evident, in various embodiments, the functionalized PPF polymers of the present invention may also contain terminal functional groups added via the starting alcohol as described above. Thus, in one or more embodiments, the functionalized PPF polymer of the present invention may have the formula:

Wherein n is an integer from 1 to 100; R is an alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, tertiary halogen group and poly (ethylene glycol) group, lactone group, and non A functional group comprising a group selected from a functional initiating alcohol moiety; R 'is an alkyl or aryl group having a functional group or a functional group which can be introduced into a click or other reaction with a corresponding functional group, among which an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, a thiol group , Halide groups, or hydroxyl groups. As set forth above, the term “protected hydroxyl group” refers to the occurrence of the desired reaction in which the protecting group is replaced with a proton to replace the hydrogen atom with a protecting group to prevent unwanted reactions until the hydroxyl group is reformed. Refer to hydroxyl group. Any suitable protecting group or protecting groups known in the art can be used, including but not limited to tert -butyloxycarbonyl (BOC) groups, trimethylsilyl ether (TMS) groups, tert -butyldimethylsilyl ether (TBDMS), or Fluorenylmethyloxycarbonyl (FMOC) groups. In some of these embodiments, n is about 5 to about 100, in other embodiments, 15 to 100, in other embodiments, 25 to 100, in other embodiments, 40 to 100, in other embodiments, 1 to 1 80, in other embodiments, from 1 to 70, in other embodiments, from 1 to 60, and in other embodiments, may be an integer from 1 to 40.

In some embodiments, the functionalized PPF polymer of the present invention may have the formula:

Wherein n is an integer from about 1 to about 100. In some of these embodiments, n is about 5 to about 100, in other embodiments, 15 to 100, in other embodiments, 25 to 100, in other embodiments, 40 to 100, in other embodiments, 1 to 1 80, in other embodiments, from 1 to 70, in other embodiments, from 1 to 60, and in other embodiments, may be an integer from 1 to 40.

In some other embodiments, the functionalized poly (propylene fumarate) polymer of the present invention can have the formula:

Wherein n is an integer from about 1 to about 100. In some of these embodiments, n is about 5 to about 100, in other embodiments, 15 to 100, in other embodiments, 25 to 100, in other embodiments, 40 to 100, in other embodiments, 1 to 1 80, in other embodiments, from 1 to 70, in other embodiments, from 1 to 60, and in other embodiments, may be an integer from 1 to 40.

In some other embodiments, the functionalized poly (propylene fumarate) polymer of the present invention can have the formula:

Wherein n is an integer from about 1 to about 100. In some of these embodiments, n is about 5 to about 100, in other embodiments, 15 to 100, in other embodiments, 25 to 100, in other embodiments, 40 to 100, in other embodiments, 1 to 1 80, in other embodiments, from 1 to 70, in other embodiments, from 1 to 60, and in other embodiments, may be an integer from 1 to 40.

In various embodiments, the monomeric functionalized PPF polymer of the invention has a number average molecular weight ( M _n ) of about 0.7 kDa to about 100,000 kDa, and a height of about 1.01 to about 1.8, as measured by size exclusion chromatography (SEC). Will have a dispersion index ( Ð _M ). In some embodiments, the monomer functionalized PPF polymer has at least 1.0 kDa, in other embodiments, at least about 5 kDa, in other embodiments, at least about 8 kDa, in other embodiments, at least about 10 kDa, in other embodiments , At least about 1000 kDa, and in other embodiments, will have a M _{n of} at least about 10,000 kDa. In some embodiments, the monomer functionalized PPF polymer is 90,000 kDa or less, in other embodiments, about 80,000 kDa or less, in other embodiments, about 70,000 kDa or less, in other embodiments, about 60,000 kDa or less, in other embodiments , Up to about 50,000 kDa, and in other embodiments, will have an M _{n of up} to about 40,000 kDa.

In some embodiments, the monomer functionalized PPF polymer has at least 1.03, in other embodiments, at least about 1.05, in other embodiments, at least about 1.10, in other embodiments, at least about 1.20, in other embodiments, at least about 1.30. , In other embodiments, it will have a polydispersity index ( Ð _M ) of about 1.40 or greater. In some embodiments, the monomer functionalized PPF polymer is at most 1.70, in other embodiments, at most about 1.60, in other embodiments, at most about 1.50, in other embodiments, at most about 1.40, in other embodiments, at most about 1.30 , And in other embodiments, will have a Ð _M of about 1.20 or less.

Functionalized Propylene Oxide Monomer

In a third aspect, the present invention relates to novel functionalized comonomers used to form the novel functionalized PPF polymers discussed above. In various embodiments, the functionalized monomers for forming the functionalized poly (propylene fumarate) polymers of the present invention are linked via ether bonds to alkyl or aryl groups having functional groups that can be introduced into the click reaction with the corresponding functional groups. And glycidyl groups to be linked. In some of these embodiments, the alkyl or aryl group will comprise an alkyne group, alkene group, hydroxyl group, protected hydroxyl group, thiol group, or halide group. In some other embodiments, the functional group can be directly linked to a glycidyl group.

In some embodiments, the novel functionalized comonomers of the invention may have the formula:

Wherein R is combined with a functional group or corresponding functional group on a bioactive compound (ie, a bioactive drug, peptide, protein, sugar, etc., as described above), functional species, or other compounds that are added to the PPF polymer Click "or other alkyl or aryl group containing functional groups that can be introduced into the reaction. In some embodiments, the targeted bioactive compound can be functionalized to add a corresponding functional group, provided that doing so does not denature or otherwise bioactive compound, functional species, or other The compound should not be added to the PPF polymer ineffectively for its intended purpose. In one or more embodiments, R will be an alkyl or aryl group containing an alkyne, alkene, hydroxyl, protected hydroxyl, thiol or halide functional group.

In at least one embodiment, the functionalized monomers of the invention are glycidyl propargyl ethers. In some embodiments, functionalized monomers of the invention may have the following formula:

In at least one other embodiment, the functionalized monomers of the invention are 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO). In some embodiments, functionalized monomers of the invention may have the following formula:

In some other embodiments, the functionalized propylene oxide can be (±) -epichlorohydrin. In some embodiments, functionalized monomers of the invention may have the following formula:

With attachment Functionalized PPF Polymer

In another aspect, the invention relates to a functionalized PPF polymer as described above further comprising one or more bioactive or other functional species attached to a functional group on the PPF polymer as described above. In one or more embodiments, the present invention relates to short chain peptides, dyes or other bioactive compounds or functional species shown in Scheme 3 and / or attached to the functionalized PPF polymer of the invention described in Examples 13 and 14 below. It may include. In some of these embodiments, the azide functionalized dye, bioactive compound or other functional species is dissolved in a suitable solvent such as an isopropyl alcohol / H ₂ O mixture and combined with a CuSO ₄ catalyst and sodium ascorbate And react with terminal or monomeric functionalized PPF polymer having alkyne functionality for about 1 hour. In these embodiments, the functionalized dyes, bioactive compounds or other functional species are added to the terminal or monomer functionalized PPF polymer via a copper assisted 1,3 huegen cyclization via a click reaction. Terminal or monomer functionalized PPF polymers with attached dyes, bioactive compounds or other functional species are then washed with isopropyl alcohol and H ₂ O to remove any non-tethered dye, bioactive Remove the compound or other functional species and catalyst.

In some other embodiments, the azide functionalized peptides can be synthesized as follows. First, the desired peptide can be synthesized by microwave-assisted solid phase peptide synthesis (SPSS) on a CEM Liberty1 peptide synthesizer using standard Fmoc chemical conditions and Wang resin. The peptide (still on the Wang resin) is then combined with bromohexanoic acid (1 mmol), diisopropylcarbodiimide (DIC) and hydroxybenzotriazole and reacted for about 2 hours to bring the Br-functionalized peptide , Which is then cut using conventional methods. The solid Br-functionalized peptide is then purified and then redissolved in 10% ethanol solution in water. The addition of the azide functional group was performed by addition of NaN ₃ and 18-crown-6 and the solution was allowed to react for about 12 hours to yield the azide functionalized peptide.

Functionalized PPF Scaffolds or Other Polymer Structures

In another aspect, the present invention relates to a 3D printed scaffold or other structure comprising a functionalized PPF polymer as described above. In one or more of these embodiments, the functionalized PPF polymer of the present invention is formed of 3-D printable resin. In some of these embodiments, 3-D printable resins are described in Luo, Y .; Dolder, CK; Walker, JM; Mishra, R .; Dean, D .; Becker, ML, Biomacromolecules , 2016 , 17 , 690-697, the disclosure of which is incorporated herein in its entirety. In these embodiments, the terminal or monomeric functionalized PPF is dissolved in equivalent mass of diethyl fumarate (DEF) and the mixture of photoinitiator and light scattering agent is mixed evenly throughout the resin. The resin is then 3-D printed and photo-crosslinked with a cDLP printer or other suitable 3-D printer to form scaffolds or other polymeric structures with available functional groups for the addition of bioactive compounds or other functional species. .

In another aspect, the present invention relates to a 3D printed scaffold or other polymeric structure comprising a functionalized PPF polymer as described above and a plurality of bioactive or other functional species, wherein the plurality of bioactive or Other functional species were attached to available functional groups on the PPF polymer after formation of the scaffold or other structure.

Process for Making End-functionalized PPF Polymer

In a second aspect, the invention also relates to a method for forming the terminal functionalized PPF polymer described above. This method is very simple. Firstly, the starting alcohol, magnesium catalyst, preferably Mg (BHT) ₂ (THF) ₂ or Mg (OEt) ₂ , maleic anhydride, and propylene oxide are placed in ampoules or other suitable sealed, dry containers and suitable solvents such as monomer (maleic anhydride and propylene oxide) concentrations of about 0.5 M to about 5 M Dissolved in toluene or hexane. As is apparent, it is important that the reaction occurs in a sealed, dry environment to avoid undesirable side reactions. In some embodiments, all of the reagents are added under an inert atmosphere and preferably under an N ₂ blanket.

As set forth above, in one or more of these embodiments of the present invention, the functionalized starting alcohol will comprise a hydroxyl group, which initiates a ring-opening polymerization reaction of maleic anhydride and propylene oxide monomer, and a functional end group. And is favorable for the post-polymerization reaction and withstands the polymerization (step 1) and the isomerization reaction (step 2). Suitable functionalized starting alcohols include, but are not limited to, propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropan-2-one, 5-hydroxy Oxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, PEG diol, α -Bromoisobutyryl 4-methanol benzylmethanoate, linear polymers having hydroxyl end groups, or combinations thereof.

The catalyst for ROP polymerization of maleic anhydride and propylene oxide may be an organometallic catalyst, preferably a magnesium catalyst. Suitable magnesium catalysts may include Mg (BHT) ₂ (THF) ₂ and Mg (OEt) ₂ . In one or more embodiments, the catalyst is Mg (BHT) ₂ (THF) ₂ . Suitable solvents for this reaction are generally nonpolar alkyl carbon chains and may include, but are not limited to, pentane, hexane, mixed hexane, heptane, octane, nonane, decane dodecane, toluene, dioxane, or combinations thereof. have. In some embodiments, the solvent is toluene. In some other embodiments, the solvent is hexane or mixed hexanes.

As indicated above, the monomer (maleic anhydride and (functionalized and / or nonfunctionalized) propylene oxide) concentration in the solution will be from about 0.5 M to about 5 M. As will be appreciated by those skilled in the art, higher monomer concentrations will result in faster conversion of monomers and will require less solvent. In some embodiments, the monomer concentration in the solution is about 0.5 M to about 4 M, in other embodiments, about 0.5 M to about 3 M, in other embodiments, about 0.5 M to about 2 M, in other embodiments, about 1 M to about 5 M, in other embodiments, from about 1.5 M to about 5 M, in other embodiments, from about 2 M to about 5 M, and in other embodiments, from about 2.5 M to about 5 M.

In these embodiments, the molar ratio of starting alcohol to magnesium catalyst is from about about 1: 1 to about 1: 1000 and the molar ratio of starting alcohol to monomer is from about 1: 5 to about 1: 1000. In some embodiments, the molar ratio of starting alcohol to magnesium catalyst is about 1: 1 to 1: 500, in other embodiments, 1: 1 to 1: 300, in other embodiments, 1: 1 to 1: 200, other In embodiments, 1: 1 to 1: 100, in other embodiments 1: 1 to 1:75, in other embodiments, 1: 1 to about 1:50, in other embodiments, from about 1: 5 to about 1:20, in other embodiments, from about 1: 5 to about 1:25, in other embodiments, from about 1: 1 to about 1:15, in other embodiments, from 1: 10 to 1: 1000, in other embodiments In the range from 1:50 to 1: 1000, in other embodiments, from 1: 100 to 1: 1000, and in other embodiments, from 1: 200 to 1: 1000. In some embodiments, the molar ratio of starting alcohol to monomer is from about 1: 5 to about 1: 500 in any portion, in other embodiments, from about 1: 5 to about 1: 300, and in other embodiments, about 1: 5 to about 1: 250, in other embodiments, about 1: 5 to about 1: 150, in other embodiments, about 1: 5 to about 1:50, and in other embodiments, about 1: 5 to about 1: 10, in other embodiments, from about 1:25 to about 1: 1000, in other embodiments, from about 1: 100 to about 1: 1000, and in other embodiments, from about 1: 200 to about 1: 1000.

As will be apparent to those skilled in the art, maleic anhydride and propylene oxide should be present in the solution in a 1: 1 molar ratio to prevent monomer consumption. It is to be understood that maleic anhydride or propylene oxide will be homopolymerized under these reaction conditions.

The ampoule or other vessel is then sealed and the solution heated to a temperature of about 40 ° C. to about 100 ° C. for about 1 h to about 96 h initiated by the starting alcohol and catalyzed by a magnesium catalyst between maleic anhydride and propylene oxide. And / or maintain the ring-opening polymerization reaction of to form the terminal functionalized PPM polymer intermediate. In some embodiments, the solution has about 40 ° C. to about 90 ° C., in other embodiments, about 40 ° C. to about 70 ° C., in other embodiments, about 40 ° C. to about 50 ° C., in other embodiments, from about 45 ° C. to In another embodiment, in another embodiment, from about 60 ° C. to about 100 ° C., and in other embodiments, it is heated to a temperature of about 70 ° C. to about 100 ° C. to form a terminal functionalized PPM polymer intermediate.

In some embodiments, the solution has about 1 h to about 6 h, in other embodiments, from about 1 h to about 12 h, in other embodiments, from about 1 h to about 24 h, in other embodiments, from about 1 h to From about 1 h to about 72 h, and in other embodiments, from about 1 h to about 96 h to generate the PPM intermediate. (See Figure 1-3)

In some embodiments, all of the maleic anhydride, propylene oxide, starting alcohol and Mg (BHT) ₂ (THF) ₂ catalyst are dissolved in toluene under a nitrogen blanket and then at a temperature of about 80 ° C. for about 1 to about 30 hours. Heating produces a PPM intermediate. In some of these embodiments, the reaction time is about 6 hours to about 24 hours, in other embodiments, from about 12 hours to about 24 hours, in other embodiments, from about 18 hours to about 24 hours, in other embodiments, From about 20 hours to about 24 hours, in other embodiments, from about 1 hour to about 24 hours, in other embodiments, from about 1 hour to about 20 hours, and in other embodiments, from about 1 hour to about 18 hours.

In some other embodiments, all maleic anhydride, propylene oxide, starting alcohol, and Mg (BHT) ₂ (THF) ₂ catalyst are all dissolved in hexane under a blanket of nitrogen and then about 45 ° C. for about 1 h to about 100 h hours. Heated to a temperature of to produce a PPM intermediate. In some of these embodiments, the reaction time is about 12 hours to about 96 hours, in other embodiments, from about 24 hours to about 96 hours, in other embodiments, from about 36 hours to about 96 hours, in other embodiments, From about 48 hours to about 96 hours, in other embodiments, from about 60 hours to about 96 hours, in other embodiments, from about 72 hours to about 96 hours, in other embodiments, from about 84 hours to about 96 hours, in other embodiments In about 90 hours to about 100 hours, in other embodiments, from about 1 hour to about 90 hours, in other embodiments, from about 1 hour to about 80 hours, in other embodiments from about 1 hour to about 70 hours . While such systems require longer polymerization times as a result of reduced temperatures, this method has been found to be advantageous with regard to scale-up reactions. The solubility of all reagents other than the polymer into hexane means that the majority of impurities are decanted from the hexane solution after polymerization and purely removed from the polymer, thus reducing the number of precipitates required to recover the pure PPM polymer intermediate. . Advantageously, when the decanted solution is cooled to room temperature, unreacted MAn recrystallizes and can be recovered for further use.

PPM polymer intermediates can be collected and purified using any known method. Those skilled in the art can use conventional techniques to collect and purify PPM polymers without undue experimentation. In one or more embodiments, the obtained PPM polymer intermediate can be recovered by repeated precipitation with an immiscible solvent such as excess diethyl ether, hexane, hexane, heptane, octane or chloroform.

In the second step, the PPM polymer intermediate is isomerized to its trans- isomer form (PPF) using any method known in the art for this purpose. In one or more embodiments, PPM polymers can be isomerized using the methods described in US Published Application No. 2016/0237212, the disclosure of which is incorporated herein by reference in its entirety. The isomerization step causes some other changes to the polymer, while the most common aspects of terminally functionalized PPF polymers of embodiments of the present invention, such as the approximate M _n , Ð _M , and T _g ranges, are determined in the first reaction. It is obvious.

In one or more of these embodiments, the PPM polymer intermediate is then placed in a suitable container such as a round bottom flask and placed in a suitable solvent such as chloroform, tetrahydrofuran (THF), dioxane, diethyl ether, or a combination thereof under an inert atmosphere. Dissolves. It is envisioned that any solvent selected can be removed without undue difficulty or cost, and in some embodiments, the solvent is chloroform. If the PPM polymer intermediate is dissolved, a catalyst, preferably diethylamine, is added. The container is then connected to a condenser and then heated to a reaction temperature of about 5 ° C to about 80 ° C. In some embodiments, the reaction temperature can be about 55 ° C to about 65 ° C. In these embodiments, the solution is heated for about 5 to about 100 hours. In some embodiments, the solution is heated for about 24 to about 48 hours.

Even when relatively small amounts of PPM polymer chains remain in the PPF polymer, this will have a negative impact on the polymer's ability to crosslink, which has been found to make it unsuitable for 3D printing and other similar applications. Thus, in essence, it is important that all PPMs be converted or eliminated into PPMs. (See FIG. 4) In some embodiments, the conversion of PPM to PPF is about 96 mass percent to about 100 mass percent. In some embodiments, the conversion of PPM to PPF is about 98 mass percent to about 100 mass percent. In some embodiments, the conversion of PPM to PPF is about 99 mass percent to about 100 mass percent. In some embodiments, the PPF polymers of the present invention have ultraviolet-visible spectroscopy (UV-Vis) spectra, Fourier transform infrared spectroscopy (FTIR) spectra, proton nuclear magnetic resonance (1H NMR) spectroscopy or matrix assisted laser desorption / ionization-flight It does not contain residual PPM polymer chains as measured by timed (MALDI-TOF) mass spectroscopy.

When the isomerization reaction is complete, the terminal functionalized PPF polymer can be isolated or purified by any suitable method known in the art for this purpose. In at least one embodiment, and the terminal functionalized PPF polymer was recovered via precipitation from hexanes, and the terminal functionalized PPF polymer was subjected to washing with phosphate buffer solution (pH = 6) to completely remove diethylamine. Was isolated and purified. The solvent may be removed by rotary evaporation or any other method known in the art for this purpose.

In order to further define and reduce the embodiments of the invention carried out, the ring-opening copolymerization of maleic anhydride and propylene oxide using Mg (BHT) ₂ (THF) ₂ as catalyst with various alcohol initiators and targeted DP is investigated. It became. Reaction conditions and yields for these tests are shown in Table 1 below:

Table 1

Various alcohol Initiator  And Targeted  As catalyst with DP Mg (BHT) ₂ (THF) ₂ To  Conditions and yields of PPF polymers produced using

Initially benzyl alcohol (BnOH) was used as the primary alcohol initiator with reagents at a total concentration of 2 M in satoluene. The reaction was carried out at 80 ° C. in a sealed, dry N ₂ atmosphere (Scheme 2). The polymerization was continued for 24 hours before quenching with excess chloroform, after which the polymer was recovered from precipitation in diethyl ether.

Scheme 2 .

Ring-opening copolymerization of propylene oxide and maleic anhydride using various initiators to form poly (propylene maleate)

Monomer conversion of MAn was monitored through ¹ H NMR spectroscopy of the crude reaction mixture and comparison of monomer proton resonance ( δ = 7.01 ppm) to corresponding polymer proton resonance ( δ = 6.26 ppm). Monomer conversion of PO was not characterized as a result of the low vapor pressure and the boiling point of PO using ¹ H NMR spectroscopy resulting in unreliable integration values. ¹ H NMR spectroscopic analysis of the recovered material showed that the polymer was poly (propylene maleate) with proton resonance corresponding to the benzyl alcohol (FIG. 1) present. In particular, there was no proton resonance ( δ = 3.3-3.5 ppm) corresponding to the methylene protons observed from the homopolymer of PO. Homopolymerization of MAn was not observed during the copolymerization process, but homopolymerization of PO was previously observed as a result of higher ring-modification resulting in higher reactivity of PO and lower selectivity of the catalyst. The ratio of MAn and PO incorporated into the polymer backbone remained nearly equimolar, so it was observed that some side reactions (such as branching or crosslinking in the alkene of the incorporated MAn) occurred.

Matrix assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF MS) was used to confirm chain end-group accuracy. As expected from the alternating copolymerization system, two major distributions were observed due to the whole polymer repeating unit or half polymer repeating unit (ie one additional maleic anhydride or propylene oxide incorporated at the chain-end). End groups for the two main distributions were calculated to correspond to the BnOH initiation, and few distributions were observed. Copolymerization of MAn and PO using Mg (BHT) ₂ (THF) ₂ as catalyst will optionally be initiated from the primary alcohol source. Previous copolymerizations using organocatalysts have been obtained in unwanted side reactions involving crosslinking at the alkene bonds and the formation of unwanted hyperbranched copolymers. In addition, the MALDI-ToF mass spectrum of PPM also confirmed the lack of PO homopolymerization, and there was no continuous mass difference due to the addition of PO adjacent to another PO repeat unit. The mechanism for the copolymerization of MAn and PO can take place in a similar manner to the ROCOP of anhydrides and epoxides using a dimethyl zinc catalyst, wherein the coordination insertion of maleic anhydride can only be initiated by the alcohol chain ends, Coordination insertion of the side can be initiated solely by carboxylic acid chain ends, which leads to alternating copolymerization.

Pseudo- first kinetics of ROCOP of MAn and PO was followed by the same conditions with targeted degree of polymerization (DP) of 25 repeat units. Aliquots were withdrawn over a period of 8 hours, and monomer conversion of MAn was determined by ¹ H NMR spectroscopic analysis of the crude mixture. (FIG. 5A) The molecular weight distribution of each aliquot was determined by SEC after precipitation. (Figure 5b) monomer conversion of MAn is exhibited secondary kinetics, the rate constant (k of the radio wave _'p) are k' was observed to be _{^{p = 1.36 × 10 -5 s -1}} . As a result of the quasi- first order kinetics, it can be estimated that the number of active chains is maintained and that no termination side reactions occur throughout the polymerization. Linear molecular weight growth and low Ð _M with increasing monomer conversion were also observed for each, which provides further evidence of controlled ROCOP.

To quantify control over copolymerization kinetics, the range of DPs was targeted. PPM was synthesized by targeting DPs of 10, 25, 50 and 100 based on the ratio of initiator to comonomer (FIG. 6). The molecular weight of the final copolymer was characterized using ¹ H NMR spectroscopy and SEC. The DP of the copolymer obtained was a ratio of benzyl alcohol methylene proton resonance ( δ = 5.01 ppm) to methine proton resonance of ring-opening propylene oxide ( δ = 5.25 ppm) and alkene proton resonance of ring-opening maleic anhydride ( δ = 6.26 ppm). Calculated based on The molecular weight was observed to increase linearly with the targeted DP with the low Ð _M observed throughout.

Naturally, the benzyl chain-terminus is not an ideal end group for modification to bioactive species after polymerization or after printing. To this end, ROCOP of MAn and PO was performed under the same conditions using propargyl alcohol as the primary alcohol initiator (Scheme 3). ¹ H NMR spectroscopy analysis of the obtained PPM showed the presence of proton resonance at δ = 4.78 and 2.27 ppm, corresponding to methylene and alkyne protons of propargyl alcohol, respectively. MALDI-ToF MS further confirmed initiation from propargyl alcohol. Two distributions were observed, one main and one minor, with two corresponding molecular weights corresponding to propargyl alcohol chain-ends. As observed with benzyl alcohol initiation, the main distribution was PPM with complete repeating units, and the minority distribution presumably contained additional propylene oxide units at the chain ends. No other distribution was observed, indicating that high chain end-group accuracy is achieved.

Scheme 3

As initiator Propargyl alcohol  using Maleic acid  Of anhydride and propylene oxide From ROCOP  Dye-and Peptide - Functionalized Poly (Propylene Fumarate ) Scaffold  synthesis

ROCOP of MAn and PO was further studied using primary alcohol 4-hydroxy-2-butanone (4HB) as starting species under the same conditions. 4HB was selected as an initiator to promote functionalization after printing with amines or hydroxylamine. Initiation from 4HB was confirmed by ¹ H NMR spectroscopy of the obtained PPM material with characteristic proton resonances of acyl methylene and methyl groups observed at δ = 4.45 and 2.19 ppm, respectively. Likewise, MALDI-ToF MS was used to confirm the chain-end accuracy of the polymer. However, as a result of the required ionization energy, cleavage side reactions occurred where the end groups were cleaved to release acrolein as a byproduct. As a result, the end group showed water initiation by MALDI-ToF MS, which was not observed in ¹ H and ¹³ C NMR spectroscopy of the same material. Previous literature has shown that the presence of water in the course of polymerization poisons the catalyst rather than initiating the polymerization. Unlike the benzyl alcohol and propargyl alcohol disclosed systems, three distributions were observed, consistent with total PPM repeat units with 1, 2 or 3 additional PO repeat units. However, each distribution exhibited 4HB chain ends, so chain end accuracy was preserved despite increased preference for PO incorporation.

To produce end group modifiable PPF, isomerization of PPM should be performed without side reactions resulting in cleavage or other side reactions of the end group species. In the previously reported process (DiCiccio, AM; Coates, GW, J. Am. Chem. Soc. , 2011 , 133 , 10724-10727), the disclosure of which is incorporated herein by reference in its entirety. Thus, isomerization of benzyl alcohol initiated PPM was performed under reflux for 24 hours at a concentration of 0.5 M in CHCl ₃ using diethylamine (0.15 equiv per alken) as catalyst. The solution was washed with sodium phosphate buffer solution (3: 1 v / v) to completely remove diethylamine before the solvent was removed via rotary evaporation. ¹ H NMR spectroscopy of the recovered polymer showed a complete decrease in proton resonance due to cis -alkene protons ( δ = 6.2 ppm) and new proton resonance due to trans- alkene protons ( δ = 6.7 ppm), which The full isomerization of cis- alkene containing PPM to trans- alkene containing PPF is shown.

Further analysis of the ¹ H NMR spectrum showed that the proton resonance of each end group was retained as an ash with an integral value consistent with the polymer prior to the isomerization, thus the end group was not affected by the isomerization process. Proven. This was further confirmed by SEC analysis of the polymer before and after isomerization showing a similar molecular weight distribution.

The properties of the PPF polymers produced in these experiments are summarized in Table 2 below:

TABLE 2

Various alcohol Initiator  And Targeted  As catalyst with DP Mg (BHT) ₂ (THF) ₂ To  Properties of PPF Polymers Created Using

See also FIGS. 1-3 and 7-12.

As a comparison for the production of PPM using other organometallic catalysts at lower temperatures, the ROCOP of MAn and PO was 2M in hexane at 45 ° C. using benzyl alcohol as initiator and Mg (BHT) ₂ (THF) ₂ as catalyst. Total monomer concentration. As a result of the lower temperatures, longer polymerization times are required and thus continued for 96 hours before polymerization quenching. Unlike polymerization in toluene, MAn was not observed to dissolve prior to heating the solution. Similarly, the polymer was observed to be immiscible with the hexane solution throughout most of the polymerization, which also prevented monomer conversion from being monitored by ¹ H NMR spectroscopy. However, analysis of the obtained polymer by ¹ H NMR spectroscopy showed that PPM was synthesized with targeted DP based on the initial molar ratio of initiator to monomer (Table 3). MALDI-ToF MS further demonstrated that end group accuracy is maintained in the polymerization process without distribution due to water initiation or transesterification side reactions with water. SEC analysis of the polymer showed that the molecular weight was consistent with both theoretical M _n and M _n based on ¹ H NMR spectroscopy with low Ð _M (1.17) even with the majority of the polymers immiscible in the reaction solution.

TABLE 3

For various DP Hexane  Molecular Weight Characteristics of PPF Produced in China

See also FIGS. 4, 13-15:

As suggested above, although such systems require longer polymerization times as a result of reduced temperatures, this method may be advantageous in terms of scale-up reactions. The solubility of all reagents other than the polymer in hexane means that a large number of impurities are removed from the polymer purely by decanting the hexane solution after polymerization, thus reducing the number of precipitates to recover pure PPM. Advantageously when the decanted solution is cooled to room temperature, unreacted MAn has been observed to recrystallize and can be recovered for further use. As a result of PPM produced in chemically identical toluene and hexanes, isomerization to PPF was performed under the same conditions, resulting in complete conversion of all cis -alkenes to trans- alkenes (FIG. 4).

= 6 mm)가 프린팅되고, 표면적을 계산하였다. Chromeo^® 546-아자이드 염료는 구리-매개된 아자이드-알킨 고리화부가 (CuAAC)를 사용하여 디스크에 부착되었고, 프로파르길 알코올 말단기의 표면 농도는 감산 농도 방법(subtractive concentration method)을 통해 계산되었다. 즉, 염료 용액의 농도의 감소는 필름이 용액으로 침적된 이후에 UV/가시광 및 형광 분광법을 통해 측정되었다. 촉매의 존재 하에 촉매 없이 필름을 코팅하고, 최초 용액에 대해 비교하였다. 표면 상의 염료의 물리적 흡착은 30.0 (± 3.3) pmol.cm^-2의 CuAAC 표면 부착 농도와 비교하여 0.1 (± 0.1) pmol.cm^-2의 농도를 가지는 것으로 결정되었다. 다른 표면 작용화된 물질, 예컨대 PEG-펩타이드 하이드로겔 및 펩타이드 가교결합된 폴리(에스테르 우레아)는 증가된 세포 생존력에서의 실증 연구를 사용하여 유사한 표면 농도를 나타내었다. 아자이드-작용화된 염료의 선택적 부착은 마스크로서 작용하는 육각형 격자 투과 전자 현미경검사 (TEM) 그리드로 피복된 박막 상의 Megastokes^® 673-아자이드 염료의 CuAAC를 통해 추가로 실증되었다. 피복된 필름은 1시간 동안 이소프로필 알코올 및 물 혼합물 중의 황산구리 및 나트륨 아스코르베이트의 용액에 침지시켰다. 필름을 3회 탈이온수로 세정하고, 이미지형성을 위해 673 nm 필터가 구비된 형광 현미경 하에 배치하였다.As indicated above, for printing 3D scaffolds, the end-functionalized PPF was mixed with the resin together with the previously reported composition. Luo, Y .; Dolder, CK; Walker, JM; Mishra, R .; Dean, D .; Becker, ML, Biomacromolecules , 2016 , 17 , 690-697, the disclosure of which is incorporated herein by reference in its entirety. Briefly, the end-functionalized PPF was dissolved in equivalent mass of diethyl fumarate (DEF) and the mixture of photoinitiator and light scattering agent (4.1 wt.%) Was uniformly mixed throughout the resin. Thin films can be printed using an EnvisionTEC Micro cDLP printer. Propargyl alcohol-functionalized PPF discs (

= 6 mm) was printed and the surface area was calculated. Chromeo ^® 546-azide dye was attached to the disc using copper-mediated azide-alkyne cycloaddition (CuAAC), and the surface concentration of propargyl alcohol end groups was determined via a subtractive concentration method. Was calculated. That is, the decrease in the concentration of the dye solution was measured by UV / visible and fluorescence spectroscopy after the film was deposited into the solution. The film was coated without catalyst in the presence of a catalyst and compared against the original solution. The physical adsorption of the dye on the surface was determined to have a concentration of 0.1 (± 0.1) pmol.cm ^-2 compared to the CuAAC surface adhesion concentration of 30.0 (± 3.3) pmol.cm ^-2 . Other surface functionalized materials such as PEG-peptide hydrogels and peptide crosslinked poly (ester ureas) exhibited similar surface concentrations using empirical studies at increased cell viability. Selective attachment of azide-functionalized dyes was further demonstrated through CuAAC of Megastokes ^® 673-azide dyes on thin films coated with hexagonal lattice transmission electron microscopy (TEM) grids acting as masks. The coated film was immersed in a solution of copper sulfate and sodium ascorbate in an isopropyl alcohol and water mixture for 1 hour. The film was washed three times with deionized water and placed under a fluorescence microscope equipped with a 673 nm filter for imaging.

To confirm that the end-functionalized polymers can be induced with bioactive peptides, cell studies were performed using mouse MC3T3-E1 cells to assess whether the peptides are bioactive after surface functionalization. Peptide sequence GRGDS is an analogue of the RGD sequence which is widely used to enhance cell adhesion to the biomaterial surface. Azide-functionalized GRGDS (N ₃ -GRGDS) was synthesized via solid phase peptide synthesis for this purpose and attached to propargyl alcohol end-functionalized PPF discs using CuAAC. (See FIG. 16) Disks were also prepared without addition of N ₃ -GRGDS and without addition of copper sulfate catalyst to serve as control specimens. Circular disks were printed to a diameter of 6 mm and washed with chloroform, acetone and ethanol to remove photoinitiator by-products. After sterilization with 70% ethanol and UV light, the discs were then tested for cytotoxicity. MC3T3-E1 cells were cultured on PPF discs at 250 cells.mm ⁻² . 37 ℃ and after 48 hours incubation in 5% CO _2, was a disk to the live / dead ^® black, where viable cells can be identified by the green knife-old AM dye, an apoptotic cell is red under fluorescence microscopy It was confirmed by the ethidium homodimer dye. Disks were quantified three times with pre-cultured glass slides used as controls (FIG. 17). Disks derived from GRGDS peptides showed similar cell viability of end-functionalized PPF (no added RGD solution) and physically adsorbed end-functionalized PPF (copper added RGD solution) control disks. . Normalization of survival ratios for the glass slide control showed greater than 90% cell viability for all films. Thus, the cytotoxicity of the end-functionalized PPF was low and was directly similar to both the step-growth polymerization produced PPF and PPF produced using Mg (OEt) ₂ as the ROCOP catalyst.

Histochemical staining of actin and nuclei was also performed to visualize cell adhesion. GRGDS functionalized discs showed improved spreading after seeding with well defined actin filament formation over a larger area than all of the end-functionalized PPF controls consistent with integrin assisted attachment. Data collected from these early cellular studies remained bioactive and bioavailable when GRGDS peptides were immobilized on propargyl alcohol-initiated PPF discs, and further functionalization with other peptides or bioactive molecules is feasible. . More advanced studies of ligand, concentration, and degradation are ongoing in related preclinical models.

End group modifiable poly (propylene fumarate) (PPF) of the present invention has been synthesized through the use of ROCOP and functional primary alcohol initiators. As a result of the characteristics of the ROCOP, end group-modifiable PPFs can be prepared at any targeted molecular weight without loss of end group functionality from transesterification side reactions or cyclization in the course of polymerization or isomerization. In addition, the ability to modify the surface of the PPF scaffold after polymerization and after printing has been demonstrated by the attachment of small molecule dyes and short chain peptides. The low cytotoxicity observed in subsequent cellular assays indicated that this method of production of PPF was similar to the previous method with the potential for cell differentiation induced through peptide functionalization of the material.

Process for preparing functionalized propylene oxide monomer

In another aspect, the present invention relates to a novel method for forming the functionalized propylene oxide comonomers discussed above. In general, this reaction involves the addition of functional groups to propylene oxide comonomers using phase transfer chemistry. In this reaction, one or more alcohols, such as propargyl alcohols, containing the desired functional groups are dissolved in an aqueous solution containing a base such as NaOH or KOH, and a solution containing halogenated propylene oxide such as (±) -epichlorohi And a phase transfer agent such as tetrabutylammonium hydrogensulfate, and an organic solvent. In the course of the reaction, molecules containing functional groups are transferred from the aqueous phase to the organic phase, where they are bonded to the ends of propylene oxide by ether bonds. Thus, by forming ether bonds in the primary halide, different types of functionalized propylene oxide can be obtained and used to synthesize functionalized PPF by ring-opening copolymerization with a metal catalyst as described in detail below.

In various embodiments, the functional groups on the alcohol are in "click" or other reactions with the corresponding functional groups on the bioactive compounds (ie bioactive drugs, peptides, proteins, sugars, etc.), or any other compound added to the PPF polymer. It may be an alkyl or aryl group containing a functional group which can be introduced. In various embodiments, alcohols used to form the functionalized propylene oxide comonomers of the present invention include, but are not limited to propargyl alcohol, o -nitrobenzyl alcohol, (±) -epichlorohydrin, or Combinations.

In one or more of these embodiments, suitable bases will include, but are not limited to, NaOH or KOH. In some of these embodiments, the halogenated propylene oxide can be, but is not limited to, (±) -epichlorohydrin. In various embodiments, the phase transfer agent can be tetrabutylammonium hydrogensulfate tetrahexylammonium hydrogensulfate tetraoctylammonium hydrogensulfate tetradecylammonium hydrogensulfate. In one or more of these embodiments, suitable organic solvents may include, but are not limited to toluene, hexane, mixed hexane, heptane, octane, dioxane, pentane, nonane, decane dodecane or combinations thereof.

In one or more embodiments, the functionalized propylene oxide comonomer can be prepared according to Scheme 4:

Scheme 4

Wherein R is a "click" or other reaction with a functional group or corresponding functional group on a bioactive compound (ie bioactive drug, peptide, protein, sugar, etc.), functional species or any other compound added to the PPF polymer Alkyl or aryl groups containing functional groups which can be introduced. In some embodiments, the targeted bioactive compound can be functionalized to add a corresponding functional group, provided that doing so does not denature or otherwise bioactive compound, functional species, or other compounds. It does not add ineffectively for its intended purpose. In one or more embodiments, R will be an alkyne, alkene, hydroxyl, protected hydroxyl, thiol or halide functional group or an alkyl or aryl group containing such functional group.

In various embodiments, the method of forming a functionalized propylene oxide comonomer of the present invention comprises propargyl in an aqueous solution containing a base selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), and combinations thereof. Start by adding alcohol. In some of these embodiments, propargyl alcohol is added dropwise to an aqueous solution containing about 20 wt% to about 50 wt% NaOH at a temperature of about −10 ° C. to about 30 ° C. with stirring. In some of these embodiments, the aqueous solution is from about 20% to about 45% by weight, in other embodiments, from about 20% to about 40% by weight, in other embodiments, from about 20% to about 35% by weight, In other embodiments, from about 20% to about 30% by weight, in other embodiments, from about 30% to about 50% by weight, in other embodiments, from about 35% to about 50% by weight, in other embodiments, It contains about 40% to about 50% by weight of NaOH. In some embodiments, the aqueous solution is about −10 ° C. to about 20 ° C., in other embodiments about −10 ° C. to about 15 ° C., in other embodiments about −10 ° C. to about 10 ° C., in other embodiments about −10 ° C. To about 5 ° C., in other embodiments from about −10 ° C. to about 0 ° C., in other embodiments from about −5 ° C. to about 30 ° C., in other embodiments from about 0 ° C. to about 30 ° C., and in other embodiments about 10 ° C. It is formed at a temperature of ℃ to about 30 ℃.

Next, (±) -epichlorohydrin and tetrabutylammonium hydrogensulfate are dissolved in a suitable organic solvent such as hexane and water is added to the aqueous propargyl alcohol solution under an inert atmosphere. The temperature is returned to ambient temperature and the reaction proceeds for about 1 to about 24 hours under an inert atmosphere such as an N ₂ blanket to produce glycidyl propargyl ether. In some embodiments, the reaction is about 5 to about 24 hours, in other embodiments, from about 10 hours to about 24 hours, in other embodiments, from about 12 hours to about 24 hours, in other embodiments, from about 1 hour to From about 1 hour to about 15 hours, and in other embodiments, from about 1 hour to about 10 hours.

The reaction is then quenched and the crude product obtained is purified by any suitable means known in the art. In some embodiments, the reaction is quenched in brine. In some of these embodiments, the crude product is extracted with a suitable organic solvent and purified by column chromatography or distillation to produce purified glycidyl propargyl ether. In some of these embodiments, the extraction step is a bath to extract the crude product with dichloromethane (DCM) in 3 batches, and drying the combined organic layers over Na ₂ SO _4, and filtered with Na ₂ SO _4, and rotary evaporated This involves concentrating the product.

In some embodiments, functionalized propylene oxide comonomers can be prepared as set forth in Example 18 below.

In some other embodiments, the functionalized propylene oxide comonomer 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO) can be prepared from o -nitrobenzyl alcohol using substantially the same reaction. have. In these embodiments, o -nitrobenzyl alcohol comprises a suitable solvent such as 1,4-dioxane or THF, and a delivery agent such as tetrabutylammonium hydrogensulfate, and a base such as sodium hydroxide (NaOH) or potassium hydroxide (KOH). It is dissolved in the aqueous solution to contain. In some of these embodiments, the aqueous solution contains about 20 wt% to about 50 wt% NaOH, and in some of these embodiments the aqueous solution is about 20 wt% to about 45 wt%, in other embodiments , From about 20 wt% to about 40 wt%, in other embodiments, from about 20 wt% to about 35 wt%, in other embodiments, from about 20 wt% to about 30 wt%, in other embodiments, about 30 wt% To about 50% by weight, in other embodiments, from about 35% to about 50% by weight, and in other embodiments, from about 40% to about 50% by weight of NaO.

Next, (±) -epichlorohydrin is added to the mixture at a temperature of about −10 ° C. to about 30 ° C. with stirring. In some embodiments, (±) -epichlorohydrin is from about −10 ° C. to about 20 ° C., in other embodiments from about −10 ° C. to about 15 ° C., in other embodiments from about −10 ° C. to about 10 ° C., other From about -10 ° C to about 5 ° C in embodiments, from about -10 ° C to about 0 ° C in other embodiments, from about -5 ° C to about 30 ° C in other embodiments, from about 0 ° C to about 30 ° C in other embodiments, And in other embodiments dropwise to the mixture at a temperature of about 10 ° C to about 30 ° C.

The temperature is then returned to ambient temperature and the reaction proceeds under an inert atmosphere such as an N ₂ blanket for about 1 hour to about 96 hours (or until the reagents have fully reacted) to form 2-[[(2-nitrophenyl ) Methoxy] methyl] oxirane (NMMO). In some embodiments, the reaction is about 1 to about 72 hours, in other embodiments, from about 1 hour to about 48 hours, in other embodiments, from about 1 hour to about 24 hours, in other embodiments, from about 6 hours to In about 96 hours, in other embodiments, from about 12 hours to about 96 hours, and in other embodiments, from about 24 hours to about 72 hours.

The crude product obtained is purified by any suitable means known in the art. In some embodiments, 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO) is extracted to the organic phase by addition of an organic solvent such as diethyl ether or THF for at least 2 hours. Each time the organic phase is separated and collected. The combined organic phases are then washed twice or more with excess H ₂ O, saturated sodium bicarbonate, and saturated sodium chloride. Likewise, the organic layer is separated and collected by washing each, then combined, dried over MgSO ₄ , filtered, concentrated by rotary evaporation and purified by column chromatography or distillation to purify 2-[[(2-nitro). Phenyl) methoxy] methyl] oxirane (NMMO).

Process for preparing monomer-functionalized PPF polymer

In another aspect, the present invention relates to a novel method of forming the functionalized PPF polymer discussed above. Typically, the ROP method for PPF synthesis uses maleic anhydride and propylene oxide as comonomers, which undergo alternating ring-opening copolymerization (ROCOP) using organometallic catalysts before isomerization to form PPF. Under corrected reaction conditions, PPF synthesis by alternating ring-opening copolymerization (ROCOP) using organometallic catalysts shows high end group accuracy, precise control of molecular weight in the course of polymerization, linear molecular weight growth and narrow monomodal molecular weight distribution. By using the functionalized propylene oxide described above in this ROP reaction, a monomer functionalized PPF polymer with functionalized side chains is produced. In one or more embodiments, these monomer functionalized PPF polymers may also be terminally functionalized through the functionalized starting alcohol as described above.

There are two features necessary for controlled polymerization, one without termination and the other without chain transfer. These two features result in ideally controlled polymerization for the synthesis of well defined molecular weight polymers. Typically, for cyclic polyesters, a common method of using controlled polymerization is to use a catalyst to initiate ROP. However, in order to prepare PPF for bone tissue engineering clinical scaffolds or any other biocompatible products, the in vivo effects of the materials should be considered. Ideal materials for bone tissue engineering are manufactured using moderate processes, processed in a non-toxic manner, and finally printed into a non-toxic scaffold. For the synthetic step, all reagents added to the reaction system must be non-toxic or easily removed in subsequent processes. Based on the current ROP method used for PPF synthesis, the catalyst and solvent alone have the potential to introduce toxicity to the final polymer. The solvent can be easily removed, and therefore the catalyst alone must be specially considered. Organometallic compounds should be low toxicity because no complete method exists for removing trace amounts from the material. To meet this requirement, it is necessary to consider both the central metal and the surrounding ligands for use in the human body.

One example of an organometallic catalyst for ROP that has been found to meet all these requirements is magnesium 2,6- di -tert-butyl-4-methylphenoxide (Mg (BHT) ₂ (THF) ₂ ). The central magnesium ion is a non-cytotoxic component to the human body, and the ligand 2,6- di- tert - butyl-4-methylphenoxide (butylated hydroxytoluene, or BHT) was introduced to the Food and Drug Administration in 1954. Has been approved for use as food additives and stabilizers and has the following general structure:

These magnesium catalysts are not only toxic but have a good ability to control the ROP and ROCOP processes, are not sensitive to air, and allow the use of different initiators without affecting catalytic activity.

In various embodiments, the functionalized PPF polymers of the invention can be synthesized as shown in Scheme 5:

Scheme 5

Wherein R is introduced into the “click” or other reaction with a functional group, or a bioactive compound (ie bioactive drug, peptide, protein, sugar, etc.) added to the PPF polymer, or a corresponding functional group on any other compound Alkyl or aryl groups containing possible functional groups; R 'is a terminal functional group as described above. In various embodiments, R ′ is, but is not limited to, one or more benzyl groups, alkyne groups, propargyl groups, allyl groups, alkenes groups, 4-dibenzocyclooctyne groups, cyclooctyne groups, ketone groups, aldehyde groups, tertiary Halogen groups, or a combination thereof.

In various embodiments, the functionalized PPF polymer of the present invention can be synthesized as shown in Scheme 6:

Scheme 6

Wherein R is a “click” or other reaction with a functional group or corresponding functional group on a bioactive compound (ie, bioactive drug, peptide, protein, sugar, etc.), functional species, or other compound added to the PPF polymer Alkyl or aryl groups containing functional groups that can be introduced into.

In one or more embodiments of the process of the invention, the functionalized propylene oxide as described above is first produced. Next, functionalized propylene oxide, starting alcohols as described above, such as benzyl alcohol, propargyl alcohol, 4-hydroxybutan-2-one, or 5-norbornene-2-ol, metal catalysts such as Mg (BHT) ₂ (THF) ₂ , and maleic anhydride are dissolved in a suitable solvent such as toluene or hexane using standard Schlenk line techniques. In various embodiments, the starting alcohol may or may not be a functionalized starting alcohol. In some of these embodiments, the total monomer concentration of the solution is from about 1M to about 6M, in other embodiments, from about 1M to about 5M, in other embodiments, from about 1M to about 4M, in other embodiments, from about 1M to About 3M, in other embodiments, from about 2M to about 6M, in other embodiments, from about 3M to about 6M, and in other embodiments, from about 4M to about 6M.

In various embodiments, the functionalized propylene oxide will comprise from about 30 mole percent to about 50 mole percent (ie, the total combined moles of functionalized propylene oxide and propylene oxide) of the total propylene oxide monomers used. In some embodiments, the functionalized propylene oxide is from about 35 mol% to about 50 mol% of the total monomers of the total propylene oxide monomers used (ie, the total combined moles of functionalized propylene and propylene oxide), in other embodiments From about 40 mol% to about 50 mol%, in other embodiments from about 45 mol% to about 50 mol%, in other embodiments from about 30 mol% to about 45 mol%, in other embodiments from about 30 mol% to about 40 Mole%, and in other embodiments from about 30 mole% to about 35 mole%. Maleic anhydride will comprise about 50 mole percent of the total monomers used (ie, the total combined moles of functionalized propylene oxide, propylene oxide and maleic anhydride).

In one or more of these embodiments, the reaction vessel is sealed and heated to a temperature of about 40 ° C. to about 80 ° C. for about 1 to about 48 hours (or until essentially all monomers are consumed) to functionalize the present invention. Cis isomer (functionalized poly (propylene maleate)) intermediate of the modified PPF polymer. In some embodiments, the reaction vessel is from about 40 ° C. to about 75 ° C., in other embodiments, from about 40 ° C. to about 70 ° C., in other embodiments, from about 40 ° C. to about 65 ° C., in other embodiments, about 40 ° C. To about 60 ° C., in other embodiments, from about 50 ° C. to about 80 ° C., in other embodiments, from about 55 ° C. to about 80 ° C., and in other embodiments, to a temperature from about 60 ° C. to about 80 ° C. In some embodiments, the reaction vessel is about 1 hour to about 50 hours, in other embodiments, from about 1 hour to about 36 hours, in other embodiments, from about 1 hour to about 30 hours, in other embodiments, about 1 hour To about 24 hours, in other embodiments, from about 6 hours to about 48 hours, in other embodiments, from about 12 hours to about 48 hours, and in other embodiments, from about 24 hours to about 48 hours. The poly (propylene maleate) intermediate may be recovered by any suitable method known in the art for this purpose. In some of these embodiments, the functionalized poly (propylene maleate) intermediate is recovered by precipitation in a suitable organic solvent such as diethyl ether.

In one or more of these embodiments, this functionalized poly (propylene maleate) intermediate is then isomerized to form the functionalized PPF polymer of the present invention. In these embodiments, the recovered functionalized poly (propylene maleate) intermediate is then dissolved in a suitable organic solvent, preferably chloroform, and an organic base such as diethylamine or pyridine is added. The resulting solution is then heated to reflux temperature in an inert atmosphere for about 1 to about 50 hours (or until substantially all of the poly (propylene maleate) intermediate is isomerized) to produce the functionalized PPF polymer of the present invention. do. In some embodiments, the solution has about 1 hour to about 36 hours, in other embodiments, from about 1 hour to about 30 hours, in other embodiments, from about 1 hour to about 24 hours, in other embodiments, from about 6 hours to About 48 hours, in other embodiments, from about 12 hours to about 48 hours, in other embodiments, from about 18 hours to about 48 hours, in other embodiments, from about 24 hours to about 48 hours, and in other embodiments, about Reflux for 36 to about 48 hours to produce the functionalized PPF polymer of the present invention.

The functionalized PPF polymer can be purified by any suitable method known in the art for this purpose. In some of these embodiments, the functionalized PPF polymer is purified by repeated washing in an excess of phosphate buffered saline solution or a suitable acid solution, combining the organic layers as described above, and drying the resulting polymer in vacuo. To produce a purified polymer.

In order to further define and reduce the embodiments of the invention to be carried out, three different “click” reactive functional groups were selected to functionalize the monomer precursors and were prepared as set forth in the Examples section below. The first functional group was an alkyl group capable of 1,3-dipolar cycloaddition. The second one was a carbonyl group, which can undergo an oxime ligation reaction. The last one was a hydroxyl group, which could tune PPF hydrophilicity and form hydrogen bonds with the potential for further functionalization.

Characterization of functionalized propylene oxide comonomer

1. Glycidyl Propargyl Ether (GPE)

Glycidyl propargyl ether (GPE) was synthesized using a phase transfer reagent ( ie tetrabutylammonium hydrogensulfate) to transfer propargyl alcohol from H ₂ O to hexane. After purification, the pure product was characterized by ¹ H NMR spectroscopy, and the data was compared with the starting material to verify the chemical structure of the product. Comparison of ¹ H GPE NMR spectral data for GPE with that of the starting material shows that the epoxy ring and alkyne functionality remain intact (FIG. 18). Furthermore, the ¹ H- ¹ H COSY NMR spectroscopic data show that five proton resonances from the epoxy structure are coupled, resulting in a complex GoShua coupling.

2. 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO)

In these experiments, 2-[[(2-nitrophenyl) methoxy] methyl] oxirane is synthesized using NaOH as the Lewis base to form ether linkages with (±) -epichlorohydrin. After purification, the final product ¹ H NMR spectroscopy data shows that the o -nitrobenzyl group is successfully attached to the epoxide structure without ring opening or other negative side reactions (FIG. 19).

Characterization of Monomer Functionalized PPM Polymer Intermediates.

Poly (glycidyl propargyl ether- co -maleic anhydride) (poly (GPE- co- MA ))

Poly (glycidyl propargyl ether- co -maleic anhydride) was synthesized by ring-opening copolymerization of glycidyl propargyl ether and maleic anhydride. After precipitation and isomerization, the trans- batch poly (GPE- co- MA) was characterized by ¹ H and ¹³ C NMR spectroscopy and the molecular weight distribution was characterized by MALDI-ToF MS and SEC.

Based on the MALDI-ToF MS data, the regular molecular weight loss (210.10 Da) between near peaks is equal to the molecular weight of the polymer repeat unit, demonstrating that functionalized PPF is obtained; Similarly the ¹ H NMR spectrum shows six different proton resonances, corresponding to six different protons in the repeat unit (FIG. 20). The ¹³ C NMR spectrum of the PPF obtained shows carbon resonance from maleic anhydride comonomer and functionalized epoxide comonomer (FIG. 21), indicating that an alternating copolymer was obtained.

To test the control that this system can have, four different target DP polymerizations were tested based on the comonomer and catalyst feed ratios. In the same course of reaction conditions, the result is that polymerization can target specific DPs with good accuracy when the target DPs are approximately 25 (Table 4). In the meantime, if the target DP exceeds 25, the system will eventually lose control as a result of poor polymer solubility, and less than 25 target DPs can be controlled. SEC results show that even if the system loses control, the polymer dispersity of the target DP 50 is still good when compared to traditional transesterification methods. This means that the ROP using the catalyst has internal properties to precisely control the polymerization.

Table 4

To different target polymerization degrees Glycidyl Propargyl  Ether and Maleic acid  Copolymerization Of Anhydrides

2. Poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride) (poly (NMMO- co- MA ))

Poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride) synthesis was performed using a method similar to poly (GPE- co- MA). ¹ H NMR of the polymer before and after isomerization, in order to determine the degree of isomerization of the alkene group in the course of reflux in CHCl ₃ with diethylamine (0.15 mol. Equivalents per alkene). Spectra were used. As observed in the NMR spectra before and after isomerization, there is a clear change in the chemical shift of the proton resonance from the alkene group after isomerization (δ = 6.27 ppm to δ = 6.89 ppm), which is cis to trans It is associated with a change in configuration (Figure 22). This is similarly indicated during the isomerization process of poly (GPE- co- MA).

Furthermore, MALDI-ToF MS data shows high end group accuracy for poly (NMMO- co- MA) only through polymers initiated by the resulting propargyl alcohol, which means that the catalysts used are different epoxides without undesirable side reactions. That means you can tolerate Side. Equivalent mass reduction and clear ¹ H, ¹³ C NMR spectra per two adjacent peaks (307.21 Da) demonstrate the presence of a functionalized chemical structure (FIGS. 23-24).

Through SEC analysis, the number average molecular weight ( M _n ) of poly (NMMO- co- MA) was determined to be about 2.6 KDa, and the weight average molecular weight ( M _w ) was determined to be 2.7 KDa, with a dispersion degree of 1.04 ( Ð _M ). For controlled polymerization, the ideal degree of dispersion is 1 and SEC indicates that the degree of dispersion of the poly (NMMO- co- MA) is very close to the ideal degree of dispersion, which means that successful control of this polymerization is achieved.

Another complete property for NMMO and MA copolymerization, as evidenced by ¹ H NMR spectroscopic data, shows that the copolymerization of NMMO and MA shows high conversion (99%), and DP is almost identical to the target DP ( ie 25). A possible reason is that the side group nitrobenzyl alters the solubility of the polymer in the reaction solvent ( ie toluene), which is consistent with less solid precipitation during the polymerization process. If other epoxides are used, poor solubility or low boiling point of the monomer can significantly affect the rate of polymerization. For example, propylene oxide boils at 34 ° C. and consequently is removed from the solution by boiling during polymerization. (See Figure 24)

Kinetic Study of Polymerization

Pseudo-first kinetics studies were conducted to understand the copolymerization process and effects of different functionalized monomers in further copolymerization processes. Two types of epoxide monomers synthesized here have been studied, and kinetic studies indicate the difference between the copolymerization rates between GPE and NMMO.

1.Kinematic Study for Copolymerization of Glycidyl Propargyl Ether and Maleic Anhydride

For the copolymerization of GPE and MA, the rates of monomer consumption of GPE and MA were similar, indicating that these two monomers worked very well in alternating copolymerization (FIG. 25); However, when long-term polymerization is considered, the GPE propagation rate is faster than the MA propagation rate (FIG. 26), which can be explained by end group analysis of MALDI-ToF MS data. Based on the MALDI-ToF MS data, there is a small amount of polymer chains terminated with GPE, where ether attachment was removed via chain cleavage side reactions. These GPE end groups are associated with higher conversion rates and faster consumption rates for relatively long periods of time. (See Table 5)

Table 5

^One Based on H NMR spectroscopy Conversion of Glycidyl Propargyl Ether (GPE) and Maleic Anhydride (MA) and ln ([M] ₀ / [M] _t )

2. Kinetic Study on Copolymerization of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane and Maleic Anhydride

Table 6

^One H NMR Spectroscopy ^a on  Based 2-[[(2- Nitrophenyl ) Methoxy ] methyl ] Oxirane  ( NMMO ) And Maleic acid  Conversion of anhydride (MA) and ln ([M] ₀ / [M] _t )

For copolymerization of NMMO and MA, the propagation rate of NMMO is faster than the rate of MA consumption (Table 6; FIG. 27). The propagation rate of NMMO will be faster than the propagation rate of MA compared to the propagation rate of the copolymerization of GPE and MA, which results in a polymer chain with more NMMO repeat units, and the copolymerization will not be strictly alternating copolymerization. According to the end group analysis of the MALDI-ToF MS data, this phenomenon can also be explained through the majority of polymer chains having two sequential NMMOs as end groups (FIG. 24).

Discussion of Different Monomer Synthesis Processes

As set forth above, the functionalization of poly (propylene fumarate) via monomer functionalization has been successfully realized. Based on the blueprint for small molecule attachment via etherification reactions, the synthesis of carbonyl functionalized epoxides has been attempted, since the carbonyl groups have the potential to undergo "click" type oxime reactions and other effective organic reactions. . Considering the chemical properties of epoxides, successful synthesis of functionalized monomers should prevent the ring opening of epoxides, and therefore a phase transfer reaction should be chosen which separates the epoxides from the active Lewis bases used to synthesize GPE. . However, it has been found that the phase transfer reaction does not work well for carbonyl for epoxide addition synthesis.

As indicated above, functionalization of PPF to alkyne and o -nitrobenzyl groups has been realized, which are two novel comonomers, glycidyl propargyl ether (GPE) and 2-[[(2-nitrophenyl), respectively. ) Methoxy] methyl] oxirane (NMMO). ¹ H and ¹ H- ¹ H COZY NMR spectral data indicate successful synthesis of both GPE and NMMO. ¹ H and ¹³ C NMR spectroscopy results demonstrate that functionalized PPF is obtained after copolymerization of maleic anhydride and functionalized comonomer, which is further supported by MALDI-ToF MS data. MALDI-ToF MS data also showed high end group accuracy for both types of functionalized PPF. SEC data indicate that the two functionalized PPF variants have low dispersion. Furthermore, based on kinetic research data, ring-opening copolymerization with magnesium catalysts is controlled polymerization without the chain transfer and termination side reactions that occur during the polymerization process. Different functionalized comonomers were observed to have different propagation rates. Functionalized PPFs can be printed using stereolithographic techniques, and their scaffolds have the ability to undergo surface modification to enhance PPF molecular interactions with cells.

Methods of Making Functionalized PPF Scaffolds or Other Structures

In another aspect, the present invention is directed to a method of making the above described functionalized PPF scaffolds and other polymer structures comprising the above described functionalized PPF polymer. As set out above, the functionalized FFP polymers of the present invention are very suitable for use in resins for 3D printing. In one or more embodiments, 3D scaffolds or other polymeric structures using resins have been formed of functionalized PPF with previously reported compositions. Luo, Y .; Dolder, CK; Walker, JM; Mishra, R .; Dean, D .; Becker, ML, Biomacromolecules , 2016 , 17 , 690-697, the disclosures of which are incorporated herein by reference in their entirety. Briefly, functionalized PPF was dissolved in the same mass of diethyl fumarate (DEF) and the mixture of photoinitiator and light scattering agent (4.1 wt.%) Was mixed uniformly throughout the resin. The resin is then printed using conventional stereolithography techniques or cDLP methods and photocrosslinked to form a functionalized PPF polymer structure.

In at least one embodiment, a PPF polymer according to the invention having a higher number average molecular weight (at least about 4000 Da) is a PPF scaffold and other polymer structures functionalized using conventional techniques such as electrospinning, extrusion, or molding. And then photochemically crosslinked. One skilled in the art will be able to construct functionalized PPF scaffolds and other polymer structures from such polymers without undue experimentation.

Methods for Attaching Functional Species to Functionalized PPF Scaffolds / Structures

In another aspect, the present invention relates to a method for attaching a bioactive material or other functional species to a functionalized PPF polymer described above. In some of these embodiments, the functional groups added to the PPF polymer as described above are selected to bind corresponding functional groups on the bioactive material or other functional species added to the polymer. As is apparent, the bioactive material or other functional species must be functionalized to have or have an available functional group that is bonded to a functional group on the PPF polymer as described above, which binds to the functionalized PPF polymer described above. Or that the functionalized to be bound should not impair or excessively impair the desirable properties or uses when the bioactive material or other functional species is attached.

As set forth above, in order to be attached, the bioactive material or other functional species must contain a functional group / moiety capable of binding to one or more functional groups on the functionalized PPF polymer of the present invention. In one or more embodiments, the bioactive material or other functional species is bound to one or more functional groups on the functionalized PPF polymer of the present invention without compromising or overly impairing desirable properties or uses when naturally attached. Will contain functional groups / moieties. As is apparent, the functional groups / moieties that can be bound to one or more functional groups on the functionalized PPF polymer of the present invention are bioactive compounds of other functional species liberated to interact with the functional groups on the functionalized PPF polymer of the present invention. It must be located in the area of Exemplary such functionalized bioactive materials or other functional species include, but are not limited to, peptides, oligomers, or proteins having cysteine residues along with available thiol groups; Peptides, oligomers, or proteins with or with a thiol functional group; Peptides, oligomers, or proteins having or functionalized with alkenes or allyl functional groups; Peptides, oligomers, or proteins having or functionalized with alkyne or propargyl functional groups; Peptides, oligomers, or proteins with or with azide functionality (see, eg, Scheme 3, Example 14); Peptides, oligomers, or proteins with or with ketone or amine functionality; Peptides, oligomers, or proteins with or functionalized with hydroxyl groups or protected hydroxyl functional groups; Peptides, oligomers, or proteins having or functionalized with cyclooctyne or 4-dibenzocyclooctyne functional groups; Alkyl or aryl groups, including fluorescent atoms or compounds functionalized with or having an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, a thiol group, or a halide group; And short-chain dyes functionalized with or having an alkyne group, alkene group, hydroxyl group, protected hydroxyl group, thiol group, or halide group (see, eg, Example 13).

However, in some other embodiments, the bioactive material or other functional species to be attached has no functional groups available or no functional groups suitable for attachment to functional groups on the functionalized PPF polymer of the present invention. In these embodiments, the bioactive material or other functional species to be attached must be functionalized or otherwise modified to include suitable functional groups. A “suitable” functional group or moiety as used herein is capable of binding with the corresponding functional group / moiety on another functionalized PPF polymer or the bioactive material / functional species to which it is attached, preferably via a click reaction. Functional groups or moieties. As will be apparent, the particular mechanism for functionalizing the bioactive material or other functional species to which it is attached depends not only on the specific material to which it is attached, but also on the range of functional groups that bind to the functional group or functional groups on the functionalized PPF polymer to which they are attached. will be. In one or more embodiments, the functional group can be danced to a bioactive material or other functional species to which it is attached as shown in Scheme 3 above. One skilled in the art will be able to attach functional groups / moieties suitable for bioactive or other functional species to which they are attached without undue experimentation.

Although not required to practice the present invention, functional groups on functionalized PPF polymers and bioactive materials or other functional species attached thereto should be selected to utilize any one of the many "click" reactions described above. As indicated above, known "click" reactions are preferred for this purpose, which are modular, extensive, stereospecific, and produce very high yields, and non-chromatographic methods such as recrystallization or distillation. This is because it uses a solvent which is insensitive to oxygen and water and, if necessary, moderate or easily removed to produce the least by-product that can be removed by and to readily use available starting materials and reagents.

Examples of click reactions that are particularly useful for this purpose include thiol reactions between thiol and alkene functional groups, thiol reactions between thiol and alkyne functional groups, 1,3-dipolar cycloaddition reactions between alkyne and azide functional groups, and And / or an oxime ligation reaction between a ketone and an amine functional group between a functional group / moiety of a bioactive or other functional species and a functional group on the functionalized PPF polymer of the invention, but many other suitable combinations are possible and present It is within the scope of the invention. For example, where the functional group / moiety of the bioactive material or other functional species is a thiol group, the functional group on the functionalized PPF polymer of the present invention may be an alkene group or an alkyne group. In this example, the bioactive material or other functional species may be attached by a thiole reaction between thiol and alkene functional groups or a thiolin reaction between thiol and alkyne functional groups. Similarly, when the functional group on the functionalized PPF polymer of the present invention is an alkyl group, a bioactive material or other functional species functionalized with or having an alkyne or azide functional group / moiety can be used. In this example, the bioactive material or other functional species can be attached by a thiolin reaction between thiol and alkyne functional groups or a 1,3-dipolar cycloaddition reaction between alkyne and azide functional groups.

In some of these embodiments, the functional groups on the PPF polymer as described above are actually protecting groups or other intermediates and must be removed or modified before the reaction to add the bioactive material or other functional species can proceed. For example, in some of these embodiments, the functional group added to the PPF polymer as described above is a halide group such as a halide group on a (±) -epichlorohydrin or a nitrobenzyl group such as 2-[[(2- Nitrophenyl) methoxy] methyl] oxirane (NMMO). In these embodiments, the nitrobenzyl groups on the NMMO are UV sensitive protecting groups, which can be easily replaced with hydroxyl groups upon exposure to specific UV wavelengths and the halide groups on (±) -epichlorohydrin are protecting groups Replaced with any suitable nucleophile. Suitable nucleophiles can include, but are not limited to, amines, alcohols, thiols and hydroxylamines.

In some embodiments, the poly (propylene fumarate) polymer of the present invention may have the following formula:

Wherein n is defined above. In one or more of these embodiments, a bioactive compound or other functional species, such as functionalized peptides, short chain peptides, proteins, short chain dyes, drugs, or markers (functionalized with azide molecules) are as described above. , 3 Huisgen cyclization may be added to the terminal propargyl functional group by click reaction.

In some embodiments, the poly (propylene fumarate) polymer of the present invention may have the following formula:

Wherein n is defined above. In these embodiments, the bioactive compound or other functional species, such as peptides, short chain peptides, proteins, short chain dyes, drugs, or markers (functionalized to include amine or hydroxylamine functional groups) are sieve base conjugation or oxime lis. It can be added to the terminal ketone functional group by a gating click reaction.

In some embodiments, the poly (propylene fumarate) polymer of the present invention may have the following formula:

Wherein n is defined above. In one or more of these embodiments, the bioactive compound or other functional species, such as peptides, short chain peptides, proteins, short chain dyes, drugs, or markers (functionalized to include thiol functional groups) is terminated by a thiole click reaction. Can be added to the functional group

In some embodiments, the functionalized PPF polymer of the present invention may have the formula:

Wherein n is an integer from about 1 to about 100. In these embodiments, bioactive compounds of other functional species such as peptides, short chain peptides, proteins, short chain dyes, drugs, or markers may be added to propargyl functional groups by thiolin click reactions.

In some other embodiments, the functionalized poly (propylene fumarate) polymer of claim 1 can have the formula:

Wherein n is an integer from about 1 to about 100. In these embodiments, the NMMO functional groups can be separated using photochemistry to yield free hydroxyl groups, which then serve as nucleophiles to react with electrophiles or other suitable groups, such as acid halide compounds or activated esters. Can be used as.

In some other embodiments, the functionalized poly (propylene fumarate) polymer of claim 1 has the formula:

Wherein n is an integer from about 1 to about 100. In these embodiments, functional species, such as any nucleophiles, can be added to the chloryl group by Sn ₂ substitution reaction. In various embodiments, suitable nucleophiles can include, but are not limited to, amines, alcohols, thiols, and hydroxylamines.

In various embodiments, the functionalized PPFs of the present invention have a low degree of dispersion, apparent end groups, and the polymerization is controlled polymerization. This property can be demonstrated by ¹ H NMR spectroscopy, ¹³ C NMR spectroscopy, SEC, MALDI-ToF mass spectroscopy and kinetic research data. Successful synthesis of functionalized PPF means that there is an opportunity to modify PPF chemical properties through functionalizing comonomers. In addition, functionalized PPF scaffolds exhibit some improved properties over PPF, and this scalable functionalization method will provide a good route for modifying PPF chemical structures for different applications.

Example

The following examples are provided to more fully illustrate the present invention, but are not to be construed as limiting its scope. In addition, some of the examples may include conclusions as to how the invention may operate, while the inventors are not intended to be bound by these conclusions, but do present a possible explanation. Furthermore, unless otherwise noted using past tense, the expressions of an example do not imply that the experiment or procedure has been performed or has not been performed, or that the results have not or have not been obtained in practice. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature), but some experimental errors and deviations may exist. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Abbreviation

BHT, 2,6-di-tert-butyl-4-methylphenoxide; cDLP, continuous digital light source processing; CuAAC, copper-assisted azide-alkyne cycloaddition; DEF, diethyl fumarate; Ð _M , dispersion; DP, degree of polymerization; DSC, differential scanning calorimetry; FDM, fusion deposition modeling; GPC, gel-penetration chromatography; I, initiator; MALDI-ToF MS, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; MAn, maleic anhydride; M _n , number average molecular weight; M _w , weight average molecular weight; NMR, nuclear magnetic resonance; PCL, poly ( ε -caprolactone); PEU, poly (ester urea); PO, propylene oxide; PLLA, poly ( L -lactic acid); PPF, poly (propylene fumarate); PPM, poly (propylene maleate); PU, poly (urethane); RI, refractive index; ROCOP, ring-opening copolymerization; ROP, ring-opening polymerization; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; THF, tetrahydrofuran; 4HB, 4-hydroxybutan-2-one.

Materials for Examples 1-17

All reagents were purchased from Millipore-Sigma except for 2,6- di -tert - 4-methylphenol purchased from Acros (Gel, Belgium). Mg (BHT) ₂ (THF) ₂ was synthesized as previously reported. Wilson, JA; Hopkins, SA; Wright, PM; Dove, AP, Polym. Chem. , 2014 , 5 , 2691-2694, the disclosure of which is incorporated herein by reference in its entirety. All solvents were purchased from Fisher, Innovative Technology Inc. Dry using Pure Solv MD-3 solvent purification system. Benzyl alcohol, propargyl alcohol, 4-hydroxybutan-2-one and propylene oxide were dried over calcium hydride for 24 hours prior to vacuum distillation. Maleic anhydride was dried in vacuo on P ₂ O ₅ for 1 week. All reagents were used as received.

Instrumental Assay for Examples 1-17

Proton ( ¹ H) NMR spectra were recorded using a Varian Mercury 300 spectrometer. Carbon ( ¹³ C) NMR spectra were recorded using a Varian NMRS 500 spectrometer. All chemical shifts were reported in parts per million (ppm) relative to the reference peak of chloroform solvent at δ = 7.26 and 77.16 ppm for ¹ H and ¹³ C NMR, respectively. Molecular weights were determined via size exclusion chromatography (SEC) using Tosoh EcoSEC HLC-8320GPC on TSKgel GMH _HR -M columns in parallel with refractive index (RI) detection. Molecular weights are calculated using a calibration curve determined from poly (styrene) standards using tetrahydrofuran (THF) with eluent flowing at a sample concentration of 1.0 mL min ⁻¹ and 10.0 mg mL ⁻¹ . MALDI-ToF mass spectra were recorded on a Bruker Ultra-Flex III MALDI-ToF / ToF mass spectrometer equipped with an Nd: YAG laser emitting at 355 nm. The instrument was operated in a cationic manner. All samples were dissolved in THF at a final concentration of 10 mg mL- ¹ . Trans- 2- [3- (4- tert -butylphenyl) -2-methyl-2-propenylidene] malononitrile (DCTB) (20 mg mL- ¹ ) served as matrix and sodium tri as a cationic agent Fluoroacetate (NaTFA) (10 mg mL- ¹ ) was prepared and mixed in a 10: 1 ratio. Matrix and sample solutions were applied on the MALDI-ToF target plate by the sandwich method. MALDI-ToF data was analyzed using FlexAnalysis software. The PPF film Envisiontec ^TM Micro Advantage Plus ^® was continuously printed by using a digital light processing (cDLP) printer. Fluorescence microscopy was performed on an Olympus IX81 fluorescence microscope equipped with FITC and TRITC filters.

Example 1

Synthesis of End-functionalized Poly (propylene Maleate) (PPM)

(General method)

Using standard Schlenk line technology, the ampoule is filled with Mg (BHT) ₂ (THF) ₂ , starting alcohol, propylene oxide and maleic anhydride. The solution was dissolved in toluene at a total monomer concentration of 2 M. The ampoule was sealed and heated to 80 ° C. for a defined period. The obtained polymer is recovered by precipitation in excess diethyl ether.

Example 2

Synthesis of End-functionalized Poly (propylene Maleate)

(General method using hexane)

Using standard Schlenk line technology, the ampoule is filled with Mg (BHT) ₂ (THF) ₂ , starting alcohol, propylene oxide and maleic anhydride. The solution was dissolved in hexane at a total monomer concentration of 2 M. The ampoule was sealed and heated to 45 ° C. for a defined period. The obtained polymer is recovered by precipitation in excess diethyl ether.

Example 3

Synthesis of Benzyl Alcohol Initiated End-functionalized Poly (propylene Maleate)

End-functionalized poly (propylene maleate) using the method set forth in Example 1 above using benzyl alcohol and magnesium catalyst as starting alcohol as shown in Scheme 7 below and using the reaction parameters shown in Table 7 below. Was synthesized.

Scheme 7

Mg (BHT) ₂ (THF) ₂ Copolymerization of Maleic Anhydride and Propylene Oxide Catalyzed by and Initiated by Benzyl Alcohol

TABLE 7

Benzyl Alcohol Initiated PPM

The presence of benzyl terminated poly (propylene maleate) product was confirmed by: ¹ H NMR (300 MHz, 303 K, DMSO- d ₆ ): δ = 7.37 (m, C ₆ H ₅ ), 6.52- 6.44 (m, OC (= 0) H = C H (= 0) O), 5.16-5.10 (m, CH ₂ C H (CH ₃ ) O and (C ₆ H ₅ ) C H ₂ O), 4.21- 4.14 (m, C H ₂ CH (CH ₃ ) O), and 1.30-1.15 (m, CH ₂ CH (C H ₃ ) O) ppm (see FIG. 1); ¹³ C NMR (125 MHz, 298 K, DMSO- d ₆ ): δ = 164.60 and 164.30 (MAn * -PO, O C OCH ₂ ), 130.58 and 130.14 (MAn * -PO, O (O) C * C H = CH), 130.00 and 129.51 (MAn * -PO, O (O) C * CH = C H), 128.40 and 128.19 (aromatic C s), 68.76 (MAn * -PO, O C H (CH ₃ ) CH ₂ ), 66.98 (MAn * -PO, OCH (CH ₃ ) C H ₂ ), 65.80 ((C ₆ H ₅ ) C H ₂ O) and 15.73 (PO, CH ₂ CH ( C H ₃ ) O) ppm; And size exclusion chromatography (SEC) (DMF): M _n = 3.7 kDa, M _w = 4.6 kDa, Ð _M = 1.3. Yield = 84%.

Example  4

Hexane  Used benzyl  Alcohol-initiated end- Functionalized Of poly (propylene maleate)  synthesis

Benzyl alcohol, hexane as starting alcohol, as shown in Scheme 8 below. And the end-functionalized poly (propylene maleate) using the magnesium catalyst and the method set forth in Example 2 above using the reaction parameters used shown in Table 8 below.

Scheme 8

Mg (BHT) ₂ (THF) ₂ Catalyzed by and initiated by benzyl alcohol

Copolymerization of Maleic Anhydride and Propylene Oxide

Table 8

Benzyl Alcohol Initiated PPM (hexane)

The presence of benzyl terminated poly (propylene maleate) product was confirmed by: ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.35 (m, C ₆ H ₅ ), 6.50-6.15 ( m, OC (= O) H = C H (= O) O), 5.25-5.16 (m, CH ₂ C H (CH ₃ ) O and (C ₆ H ₅ ) C H ₂ O), 4.24-4.12 ( m, C H ₂ CH (CH ₃ ) O), and 1.39-1.11 (m, CH ₂ CH (C H ₃ ) O) ppm. ¹³ C NMR (125 MHz, 298 K, CDCl ₃ ): δ = 164.60 and 164.30 (MAn * -PO, O C OCH ₂ ), 130.58 and 130.14 (MAn * -PO, O (O) C * C H = CH ), 130.00 and 129.51 (MAn * -PO, O (O) C * CH = C H), 128.40 and 128.19 (aromatic C s), 68.76 (MAn * -PO, O C H (CH ₃ ) CH ₂ ), 66.98 (MAn * -PO, OCH (CH ₃ ) C H ₂ ), 65.80 ((C ₆ H ₅ ) C H ₂ O) and 15.73 (PO, CH ₂ CH ( C H ₃ ) O) ppm. SEC (DMF): M _n = 3.7 kDa, M _w = 4.6 kDa, Ð _M = 1.3. (See FIG. 27)

Example 5

Synthesis of Propargyl Alcohol Initiated End-functionalized Poly (propylene Maleate)

End-functionalized polys were prepared using the method set forth in Example 1 above using propargyl alcohol and magnesium catalyst as starting alcohol as shown in Scheme 9 below and the reaction parameters used shown in Table 9 below. Propylene maleate).

Scheme 9

Catalyzed by Mg (BHT) ₂ (THF) ₂ and by propargyl alcohol

 Copolymerization of the disclosed maleic anhydride and propylene oxide

Table 9

Propargyl Alcohol Initiated PPM

Propargyl end of the presence of terminated poly (propylene maleate) of product was confirmed by the ^{following: 1 H NMR (300 MHz,} 303 K, DMSO- d 6): δ = 6.54-6.27 (m, OC (= O ) H = C H (= O) O), 5.39-5.21 (m, CH ₂ C H (CH ₃ ) O), 4.88 (s, HC≡CC H ₂ O), 4.78 (s, HC≡CC H ₂ 0), 4.38-4.21 (m, C H ₂ CH (CH ₃ ) O), 2.27 (s, H C≡C) and 1.38-1.19 (m, CH ₂ CH (C H ₃ ) O) ppm; ¹³ C NMR (125 MHz, 298 K, DMSO- d ₆ ): δ = 164.60 and 164.29 (MAn * -PO, O C OCH ₂ ), 130.58 and 130.14 (MAn * -PO, O (O) C * C H = CH), 130.00 and 129.51 (MAn * -PO, O ( O) C * CH = C H), 83.56 (HC≡ C CH 2), 68.90 (H C≡ CCH 2) 68.76 (MAn * -PO, O C H (CH ₃ ) CH ₂ ), 66.98 (MAn * -PO, OCH (CH ₃ ) C H ₂ ), 65.83 (HC≡C C H ₂ O) and 15.72 (CH ₂ CH ( C H ₃ ) O) ppm; And SEC (DMF): M _n = 3.4 kDa, M _w = 3.5 kDa, Ð _M = 1.1. Yield = 83%.

Example 6

Hexane  Used Propargyl alcohol  Disclosed terminal- Functionalized Of poly (propylene maleate)  synthesis

End-functionalized polys were prepared using the method set forth in Example 2 above using benzyl alcohol, hexane and magnesium catalysts as starting alcohols as shown in Scheme 10 below and the reaction parameters used shown in Table 10 below. Propylene maleate).

Scheme 10

Mg (BHT) ₂ (THF) ₂ on  due to Being catalyzed , Propargyl alcohol  Initiated by Maleic acid  Copolymerization Of Anhydride And Propylene Oxide

Propargyl end of the presence of terminated poly (propylene maleate) of product was confirmed by the ^{following: 1 H NMR (300 MHz,} 303 K, DMSO- d 6): δ = 6.54-6.27 (m, OC (= O ) H = C H (= O) O), 5.39-5.21 (m, CH ₂ C H (CH ₃ ) O), 4.88 (s, HC≡CC H ₂ O), 4.78 (s, HC≡CC H ₂ 0), 4.38-4.21 (m, C H ₂ CH (CH ₃ ) O), 2.27 (s, H C≡C) and 1.38-1.19 (m, CH ₂ CH (C H ₃ ) O) ppm. ¹³ C NMR (125 MHz, 298 K, DMSO- d ₆ ): δ = 164.60 and 164.29 (MAn * -PO, O C OCH ₂ ), 130.58 and 130.14 (MAn * -PO, O (O) C * C H = CH), 130.00 and 129.51 (MAn * -PO, O ( O) C * CH = C H), 83.56 (HC≡ C CH 2), 68.90 (H C ≡CCH 2) 68.76 (MAn * -PO, O C H (CH ₃ ) CH ₂ ), 66.98 (MAn * -PO, OCH (CH ₃ ) C H ₂ ), 65.83 (HC≡C C H ₂ O) and 15.72 (CH ₂ CH ( C H ₃ ) O) ppm. SEC (DMF): M _n = 750 Da, M _w = 1170 Da, Ð _M = 1.55. (Reference figure 1)

Example  7

Synthesis of 4-hydroxybutan-2-one Initiated End-functionalized Poly (propylene Maleate)

End-action using the method set forth in Example 1 using 4-hydroxybutan-2-one and magnesium catalyst as starting alcohol as shown in Scheme 11 below and the reaction parameters used shown in Table 10 below. Synthesized poly (propylene maleate) was synthesized.

Scheme 11

Mg (BHT) ₂ (THF) ₂ Copolymerization of propylene oxide and maleic anhydride initiated by 4-hydroxybutan-2-one, catalyzed by

Table 10

4-hydroxybutan-2-one initiated PPM

The presence of 4-hydroxybutan-2-one terminated poly (propylene maleate) product was confirmed by: ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 6.42-6.28 (m, OC (= O) H = C H (= O) O), 5.33-5.25 (m, CH ₂ C H (CH ₃ ) O), 4.32-4.18 (m, C H ₂ CH (CH ₃ ) O), 4.08 (m, CH ₂ C H ₂ O), 3.64 and 3.53 (m, CH ₃ C (= 0) C H ₂ CH ₂ ), and 1.38-1.13 (m, CH ₂ CH (C H ₃ ) O and C H ₃ C (═O) CH ₂ ) ppm (see FIG. 3); ¹³ C NMR (125 MHz, 303 K, DMSO- d ₆ ): δ = 165.42 (CH ₃ C (= O) CH ₂ ), 164.60 and 164.30 (MAn * -PO, O C OCH ₂ ), 130.55 and 130.16 ( MAn * -PO, O (O) C * C H = CH), 129.81 and 129.84 (MAn * -PO, O (O) C * CH = C H), 128.15, 68.77 (MAn * -PO, O C H (CH ₃ ) CH ₂ ), 66.97 (MAn * -PO, OCH (CH ₃ ) C H ₂ ), 65.81 (CH ₂ C H ₂ O), 30.63 (C (= O) C H ₂ CH ₂ ), 25.08 ( C H ₃ C (= 0) CH ₂ ) and 15.74 (CH ₂ CH ( C H ₃ ) O) ppm; And SEC (THF): M _n = 1.5 kDa, M _w = 2.0 kDa, Ð _M = 1.3. Yield = 88%.

Example 8

General Procedure for Isomerization of PPM

End-functionalized poly (propylene fumarate) was dissolved in chloroform. Diethylamine was added to the solution and refluxed for 24 hours under nitrogen atmosphere. After cooling to room temperature, the organic solution was washed with phosphate buffer solution (pH = 6) and the polymer was recovered via precipitation from hexane.

Example 9

Isomerization of PPM

End-functionalized poly (propylene maleate) (11 g, 8 mol. Eq. Olefin) was dissolved 1125 ml of chloroform. 0.01 ml of diethylamine, 0.15 mol. eq. Olefin) was added to the solution and refluxed for 24 hours under nitrogen atmosphere. After cooling to room temperature, the organic solution was washed with 1350 ml of phosphate buffer solution, pH; 6) and the PPF polymer was recovered via precipitation from hexane. (See Figure 2)

Example 10

Isomerization of Benzyl Functionalized PPM

Benzyl functionalized poly (propylene maleate) isomerized using the method shown in Examples 8 and 9 to form benzyl terminated poly (propylene fumarate) as shown in Scheme 12 below.

Scheme 12

Poly (propylene fumarate)  To form Of poly (propylene maleate) Isomerization

Isomerization of benzyl functionalized poly (propylene maleate) with the corresponding benzyl functionalized poly (propylene fumarate) was confirmed by: ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.32 (m, Ar), 6.87 (m, OC (= 0) H = C H (= 0) O), 5.36 (m, CH ₂ C H (CH ₃ ) O), 5.09 (s, C = OOC H _{2 - Ar), 4.04 (m,} C H 2 OC = O), 2.28 (s, εCL C H 2 C = OO), 1.26 (m, CH 2 CH (C H 3) O), 1.60 and 1.33 (all remaining Hydrogen) ppm.

Example 11

Isomerization of propargyl-functionalized poly (propylene fumarate)

Propargyl poly (propylene maleate) isomerized using the general method presented above to form propargyl terminated poly (propylene fumarate) as shown in Scheme 13 below.

Scheme 13

Isomerization of propargyl poly (propylene maleate) to form propargyl terminated poly (propylene fumarate) was confirmed by: ¹ H NMR (300 MHz, 303 K, DMSO- d ₆ ) : δ = 6.84-6.64 (m, OC (= O) H = C H (= O) O), 5.27-5.07 (m, CH ₂ C H (CH ₃ ) O), 4.85 (s, HC≡CC H ₂ O), 4.78 (s, HC≡CC H ₂ O), 4.44-4.14 (m, C H ₂ CH (CH ₃ ) O), 2.32 (s, H C≡C) and 1.38-1.10 (m, CH ₂ CH (C H ₃ ) O) ppm. Yield = 98%. ¹ H NMR spectral comparison of propargyl poly (propylene fumarate) (top) to precursor propargyl poly (propylene maleate) (bottom) (300 MHz, 303 K, DMSO- d- ₆ ) is shown in FIG. Is shown.

Example 12

Subtraction method of surface concentration determination

Sikyeotgo impregnated for 1 hour, the PPF thin film (1 cm × 1 cm) of the dye solution (50% v / v solution in 0.5 μM Chromeo ^® 546- azide dye, 3.2 mg sodium ascorbate in EtOH / H ₂ O), The concentration of the solution was determined using fluorescence spectroscopy. Unused solution was used as a standard to measure dye adhesion to the film.

Example 13

Propargyl Functionalized  To PPF Megastokes ^®  673- Azide  Dye Copper Mediated Azide Alkin Ring

Megastokes ^® 673-Azide Solution (50 mM 50 isopropyl alcohol: 1 mM dye, 0.5 mg CuSO ₄ , 1.5 mg sodium ascorbate in 50% v / v solution of H ₂ O) was pipetted onto PPF film, isopropyl alcohol and H _It was left for 1 hour prior to washing with ₂ O to remove any non-fixed dyes and catalysts.

Example 14

Synthesis of N3-GRGDS Peptides

GRGDS was synthesized by microwave-assisted solid phase peptide synthesis (SPSS) on a CEM Libertyl peptide synthesizer using standard Fmoc chemical conditions (0.25 mmol scale). 6-bromohexanoic acid (1 mmol) was added together with GRGDS Wang resin (0.25 mmol), diisopropylcarbodiimide (DIC, 1.1 mmol) and hydroxybenzotriazole (HOBt, 1.1 mmol) and 2 hours Reacted for a while. Br-functionalized peptides were then cleaved from the resin using a 15 mL solution of trifluoroacetic acid, triisopropylsilane, and water (95: 2.5: 2.5 vol.%). After three grinding cycles in diethyl ether, the resulting white solid was dried under vacuum overnight. The solid was then redissolved in a 10% ethanol solution in water. Coupling of the azide groups was performed after microwave synthesis. NaN ₃ (1.25 mmol) and 18-crown-6 (0.0625 mmol) were added to the Wang resin and reacted for 12 hours to yield N ₃ -GRGDS (ESI m / z: [M + H] ⁺ C ₂₃ H ₄₀ N ₁₁ O ₁₀ calcd for 630.29; measured value 630.157).

Example 15

Seeding of MC3T3-E1 on Poly (propylene Fumarate) Disc

Propargyl alcohol-initiated poly (propylene fumarate) disks (ø = 6 mm) were each rinsed with chloroform, acetone, and ethanol for 1 hour and then immersed in 1 × PBS for 12 hours. Later, the film was sterilized by immersion in 70% EtOH for 1 hour, followed by 15 minutes of exposure to UV light. Prior to cell seeding the film was immersed in Alpha-MEM for 2 hours prior to cell seeding. Mouse cranial stem cells (MC3T3-E1) were incubated in Alpha-MEM medium supplemented with 10% fetal calf serum (FBS), 100 units / mL penicillin, and 100 μg.mL- ¹ streptomycin and passaged every 3 days Incubated. MC3T3 with passage 8 was seeded at 250 cells.mm ⁻² , and all subsequent experiments were performed for 48 hours after cell seeding.

Example 16

MC3T3 Cell Viability on Poly (propylene Fumarate) Discs

Cell viability was assessed using the live / dead viability cytotoxicity kit at the 48 hour time point. Briefly, 5 μL of 4 mM calcein-AM mother liquor and 10 μL of 2 mM ethidium homodimer-1 (EthD-1) mother liquor were added to 10 mL of PBS to prepare a live / dead staining solution. Samples were washed three times with 1 x PBS (1 mL). Mother liquor (200 μL) was added to each sample and incubated for 15 minutes. The staining solution was then removed and the samples were observed under an IX81 fluorescence microscope using FITC and TRITC emission filters. For analysis, 10 random portions were selected per film and each film was run three times. Values were normalized to cell viability calculated on glass slides.

Example 17

MC3T3 Cell Dispersion on Poly (propylene Fumarate) Discs

Cell dispersion was assessed by staining cytoskeletal actin at 48 hours after cell seeding. Samples were pre-fixed in 3.7% paraformaldehyde in CS buffer solution for 1 hour, washed three times with 1 × PBS and stored at −80 ° C. until stained. For staining, samples were incubated for 10 minutes in 0.5% v / v Triton X-100 in CS buffer solution and washed three times with 1 × PBS. Next, samples were incubated for 10 minutes in 0.1 wt.% NaBH ₄ solution in 1 × PBS solution and washed three times with 1 × PBS. Rhodamine paloidine (1:40 v / v in 1 × PBS) was then added to the sample and incubated for 1 hour. After three washes with 1 × PBS, DAPI solution (6 μl of 5 μg · mL ⁻¹ DAPI in 10 mL PBS) was added to the sample and incubated for 20 minutes. After three washes with 1 x PBS, the samples were mounted using fluorescence mounting medium and imaged using a fluorescence microscope.

Materials and Devices for Examples 18-26

The materials and devices used herein are shown in Tables 11 and 12 below.

Table 11

Substances Used

Table 12

Apparatus Used in Examples 18-26

Characterization for Examples 18-26

Proton ( ¹ H) NMR spectra were recorded using a Varian Mercury-300 NMR spectrometer. Carbon ( ¹³ C) NMR spectra were recorded using a Varian Mercury-500 NMR spectrometer. All chemical shifts were reported in parts per million (ppm) for the reference peak of chloroform solvent at δ = 7.26 ppm and 77.16 ppm for ¹ H and ¹³ C NMR, respectively. Molecular weights were determined via size exclusion chromatography (SEC) using Tosoh EcoSEC HLC-8320GPC on PLgel mixed-C type columns in parallel with refractive index (RI) detection. The weight was calculated using a calibration curve determined from poly (styrene) standard with DMF (0.1% LiBr) as eluent flowing at 0.5 mL.min ⁻¹ and sample concentration 20 mg.mL ⁻¹ . MALDI-ToF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectra were recorded using a Bruker UltraFlex III MALDI Tandem Time of Flight (TOF / TOF) mass spectrometer.

Example 18

Synthesis of Glycidyl Propargyl Ether (GPE)

Glycidyl propargyl ether (GPE) was synthesized as shown in Scheme 14 below.

Scheme 14

Synthesis of Glycidyl Propargyl Ether (GPE)

Propargyl alcohol (7.6 ml, 0.135 mol) was added dropwise to 40% aqueous sodium hydroxide solution (56.5 g NaOH in 85 g H ₂ O) stirred at 0 ° C. The mixture was stirred for 30 minutes. A solution of (±) -epichlorohydrin (25 g, 0.27 mol), hexane (90 ml), tetrabutylammonium hydrogensulfate (2.29 g, 6.75 mmol) and H ₂ O (12.5 ml) was added to the reaction system It was. The reaction was allowed to warm to room temperature and the reaction was continued for 8 hours under N ₂ blanket. The reaction was quenched with brine (125 ml) and the crude product was extracted with three portions of 125 mL of dichloromethane (DCM). Layers were dried over Na ₂ S0 ₄ , filtered and concentrated by rotary evaporation. The final product was obtained by column chromatography (1: 3 hexanes / DCM to DCM), which gave GPE in 7.3 g (48.1%) yield (Scheme 3.1). ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 3.98 (t, OC H ₂ C≡CH), 3.61-3.56 (q, CHC H ₂ OCH ₂ ), 3.26-3.20 (q, CHC H ₂ OCH ₂ ), 2.95-2.89 (m, C H CH ₂ OCH ₂ ), 2.57-2.54 (t, C H ₂ (O) CHCH ₂ O), 2.39-2.35 (m, C H ₂ (O) CHCH ₂ O , C≡C H ) ppm.

Example 19

Synthesis of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO)

2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO) was synthesized as shown in Scheme 15 below.

Scheme 15

Synthesis of 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO)

o -nitrobenzyl alcohol (10 g, 0.065 mol) is dissolved in 30 mL of 1,4-dioxane, then tetrabutylammonium hydrogensulfate (1.11 g, 3.27 mmol) and 40% aqueous sodium hydroxide solution (12 g 8 g NaOH in H ₂ O) was added. (±) -Epichlorohydrin (20 mL, 0.26 mol) was then added dropwise to the mixture at 0 ° C. and the reaction was allowed to warm to room temperature. After stirring for 48 h under N ₂ , the reaction mixture was extracted with two portions of 50 mL of diethyl ether. The combined ether fractions were washed with excess water, saturated sodium bicarbonate, and saturated sodium chloride. Layers were dried over MgSO ₄ , filtered and concentrated by rotary evaporation. The final product was obtained by column chromatography (5: 3 petroleum ether / diethyl ether) to give NMMO in yield of 3.7 g (27.2%) (Scheme 3.2). ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.95-7.32 (m, Ar), 4.85 (s, ArC H ₂ OCH ₂ ), 3.84-3.79 (q, CHC H ₂ OCH ₂ ), 3.45 -3.39 (q, CHC H ₂ OCH ₂ ), 3.16-3.11 (m, C H CH ₂ OCH ₂ ), 2.74-2.71 (t, C H ₂ (O) CHCH ₂ O), 2.58-2.55 (q, C H ₂ (O) CHCH ₂ O) ppm.

Example 20

Synthesis of Magnesium 2,6- di -tert-butyl-4-methylphenoxide (Mg (BHT) ₂ (THF) ₂ )

Using standard Schlenk line technology, Schlenk is filled with 2,6- di -tert-butyl-4-methylphenol (BHT) (6.66 g, 30 mmol) and dried toluene (30) added by cannula lancer ml). Di-n- butylmagnesium (1M in hexane, 15 ml, 15 mmol) was added dropwise to the reaction with stirring. The reaction was stirred for an additional 2 hours, after which the solvent was removed. Hexane (12.5 ml) was added to the reaction vessel, followed by addition of tetrahydrofuran (THF) (2.5 ml). After stirring for 2 h under N ₂ , the solvent can be removed and the final product is obtained as a solid (Scheme 16).

Scheme 16

Magnesium 2,6-di-tert-butyl-4-methylphenoxide (Mg (BHT) ₂ (THF) ₂ ) Synthesis

Example 21

General Procedure for Polymerization of Functionalized PPF

Ampoules were purified using standard Schlenk line technology using Mg (BHT) ₂ (THF) ₂ (121.4 mg, 0.2 mmol), benzyl alcohol (0.02 mL, 0.2 mmol), epoxide (0.5 mmol) and maleic anhydride (490.3 mg , 0.5 mmol). The reagent was dissolved in toluene at the total monomer concentration of toluene. The muffle was sealed and heated to 80 ° C. for 24 hours. The obtained polymer was recovered by precipitation in excess diethyl ether. After centrifugation, the crude product was dissolved in chloroform, then diethylamine was added to the mixture, and then refluxed under N _{2 for} 24 hours. Finally, the mixture was washed with excess phosphate buffered saline solution (0.5 M). The organic layers were combined and dried to recover the polymer.

Example 22

Synthesis of Poly (Epichlorohydrin-co- maleic anhydride)

Poly (epichlorohydrin-co-maleic anhydride) was synthesized from (±) -epichlorohydrin and maleic anhydride as shown in Scheme 17 below.

Scheme 17

Copolymerization of Epichlorohydrin and Maleic Anhydride

And isomerization of poly (ECH-co-MA)

Ampoules were prepared using standard Schlenk line technology using Mg (BHT) ₂ (THF) ₂ (121.4 mg, 0.2 mmol), benzyl alcohol (0.02 mL, 0.2 mmol), (±) -epichlorohydrin (0.5 mmol) and Filled with maleic anhydride (490.3 mg, 0.5 mmol). The reagent was dissolved in toluene at a total monomer concentration of 2 M. The ampoule was sealed and heated to 80 ° C. for 24 hours. The obtained polymer was recovered by precipitation in excess diethyl ether. After centrifugation, the crude product was dissolved in chloroform, then diethylamine was added to the mixture, which was then refluxed for 24 h under N ₂ . Finally, the mixture was washed with excess phosphate buffered saline solution (0.5 M). The organic layers were combined and dried to recover the polymer.

The polymer obtained was characterized by ¹ H NMR and the results reported in Table 5 and FIG. 28 herein. ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.36 (m, Ar), 6.86 (m, CO H C = C H CO), 5.35 (m, OCH ₂ C H (CH ₂ OCH ₂ C ) CH) O), 5.22 (s, ArC H ₂ O), 4.80-4.40 (m, OCH ₂ CH (C H ₂ OCH ₂ C≡CH) O), 4.18 (m, OC H ₂ CH (CH ₂ OCH ₂ C≡CH) O), 3.74 (d, CH (CH ₂ OC H ₂ C≡CH) O), 2.49 (s, CH (CH ₂ OCH ₂ C≡C H ) O) ppm. The resulting polymer was also characterized by ¹³ C NMR and the results reported in FIG. 29 herein. ¹³ C NMR (125 MHz, 303 K, CDCl ₃ ): δ = 164.30 (O- C = O), 130.03 (H C = C H), 128.74 (Ar C ), 71.28 (OC H ₂ CH), 67.36 ( (C ₆ H ₅ ) C H ₂ O), 63.01 (CH ₂ C H (CH ₂ Cl) O), 41.89 (CH C H ₂ Cl) ppm. SEC (DMF): M _n = 6.0 kDa, M _w = 7.0 kDa, Ð _M = 1.16.

The results of Example 22 are summarized in Table 13 below.

Table 13

P (ECH-co-MA)

Example 23

Poly (glycidyl propargyl ether-co-maleic anhydride)

Poly (glycidyl propargyl ether-co-maleic anhydride) was converted to glycidyl propargyl ether (prepared as shown above in Example 18 above) and maleic anhydride as shown in Scheme 18 below. Synthesized from

Scheme 18

Copolymerization of Glycidyl Propargyl Ether and Maleic Anhydride

And isomerization of poly (GPE-co-MA)

Ampoules were prepared using standard Schlenk line technology using Mg (BHT) ₂ (THF) ₂ (121.4 mg, 0.2 mmol), benzyl alcohol (0.02 mL, 0.2 mmol), glycidyl propargyl ether (0.5 mmol) and male. Filled with acid anhydride (490.3 mg, 0.5 mmol). The reagent was dissolved in toluene at a total monomer concentration of 2 M. The ampoule was sealed and heated to 80 ° C. for 24 hours. The obtained polymer was recovered by precipitation in excess diethyl ether. After centrifugation, the crude product was dissolved in chloroform, then diethylamine was added to the mixture, which was then refluxed for 24 h under N ₂ . Finally, the mixture was washed with excess phosphate buffered saline solution (0.5 M). The organic layers were combined and dried to recover the polymer.

The resulting polymer was characterized by ¹ H NMR and the results reported in Table 14 and FIG. 31 herein. ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.36 (m, Ar), 6.86 (m, CO H C = C H CO), 5.35 (m, OCH ₂ C H (CH ₂ OCH ₂ C ) CH) O), 5.22 (s, ArC H ₂ O), 4.80-4.40 (m, OCH ₂ CH (C H ₂ OCH ₂ C≡CH) O), 4.18 (m, OC H ₂ CH (CH ₂ OCH ₂ C≡CH) O), 3.74 (d, CH (CH ₂ OC H ₂ C≡CH) O), 2.49 (s, CH (CH ₂ OCH ₂ C≡C H ) O) ppm. The resulting polymer was characterized by ¹³ C NMR and the results reported in FIG. 32 herein. ¹³ C NMR (125 MHz, 303 K, CDCl ₃ ): δ = 164.20 (O- C = O), 133.79 (H C = C H), 79.12 (H ₂ C ≡ CH), 75.57 (H ₂ C≡ C H), 71.17 (OC H ₂ CH), 67.59 ( C H ₂ OCH ₂ C≡CH), 63.51 (CH ₂ C H (CH ₂ OCH ₂ C≡CH) O), 58.76 (CH ₂ O C H ₂ C CH) ppm. The polymer was also characterized by size exclusion chromatography (SEC). SEC (DMF): M _n = 7.6 kDa, M _w = 10.6 kDa, Ð _M = 1.40. The results of Example 23 are summarized in Table 14 below.

Table 14

P (GPE-co-MA)

Example 24

Poly (glycidyl propargyl ether-co-maleic anhydride) (poly (GPE-co-MA)) ⁷ Synthesis of

Ampoules were prepared using standard Schlenk line technology using Mg (BHT) ₂ (THF) ₂ (121.4 mg, 0.2 mmol), benzyl alcohol (0.02 mL, 0.2 mmol), glycidyl propargyl ether (0.58 mL, 0.5 mmol). ) And maleic anhydride (490.3 mg, 0.5 mmol). The reagent was dissolved in toluene at a total monomer concentration of 2 M. The ampoule was sealed and heated to 80 ° C. for 24 hours. The obtained polymer was recovered by precipitation in excess diethyl ether. After centrifugation, the crude product was dissolved in chloroform, then diethylamine was added to the mixture, which was then refluxed for 24 h under N ₂ . Finally, the mixture was washed with excess phosphate buffered saline solution (0.5 M). The organic layers were combined and dried to give functionalized poly (GPE- co- MA) (Scheme 19).

Scheme 19

Poly (glycidyl propargyl ether-co-maleic anhydride)

Synthesis and Isomerization of (Poly (GPE-co-MA))

The resulting polymer was also characterized by: ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.36 (m, Ar), 6.86 (m, CO H C = C H CO), 5.35 ( m, OCH ₂ C H (CH ₂ OCH ₂ C≡CH) O), 5.22 (s, ArC H ₂ O), 4.80-4.40 (m, OCH ₂ CH (C H ₂ OCH ₂ C≡CH) O), 4.18 (m, OC H ₂ CH (CH ₂ OCH ₂ C≡CH) O), 3.74 (d, CH (CH ₂ OC H ₂ C≡CH) O), 2.49 (s, CH (CH ₂ OCH ₂ C≡) C H ) O) ppm and ¹³ C NMR (125 MHz, 303K, CDCl ₃ ): δ = 164.20 (m, O- C = O), 133.79 (m, -C = C- ), 79.12 (s, -C ≡ CH), 75.57 (s, -C≡ C H), 71.17 (s, -OC H ₂ CH-), 67.59 (s, -C H ₂ OCH ₂ C≡CH), 63.51 (s, CH ₂ C H (CH ₂ OCH ₂ C≡CH) O), 58.76 (s, -CH ₂ O C H ₂ C≡CH) ppm. (See Figures 30-32)

Example 25

Synthesis of Poly (2-[[(2-nitrophenyl) methoxyl] methyl ] oxirane-co-maleic anhydride)

2-[[(2-nitrophenyl) prepared as poly (2-[[(2-nitrophenyl) methoxyl] methyl] oxirane-co-maleic anhydride) (as set forth above in Example 19 above) ) Methoxy] methyl] oxirane (NMMO) and maleic anhydride as shown in Scheme 20 below.

Scheme 20

2-[[(2-nitrophenyl) methoxyl] methyl] oxirane

And maleic anhydride copolymerization and isomerization of poly (NMMO-co-MA)

The resulting polymer was characterized by ¹ H NMR and the results reported in Table 15, and FIG. 33. ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.98-7.38 (m, Ar), 6.27 (m, CO H C = C H CO), 5.54-5.34 (d, OCH ₂ C H (CH ₂ OCH ₂ ArNO ₂ ) O), 4.86 (s, OCH ₂ CH (CH ₂ OC H ₂ ArNO ₂ ) O), 4.41 (s, OC H ₂ CH (CH ₂ OCH ₂ ArNO ₂ ) O), 3.76 (m , OCH ₂ CH (C H ₂ OCH ₂ ArNO ₂ ) O) ppm. The resulting polymer was also characterized by ¹³ C NMR and the results reported in FIG. 34 herein. ¹³ C NMR (125 MHz, 303 K, CDCl ₃ ): δ = 163.99 (O- C = O), 147.47 (Ar C), 133.68 (H C = C H), 129.19-124.76 (m, Ar C), 71.93-71.22 (OCH ₂ CH (CH ₂ O C H ₂ Ar) O), 70.16 (O C H ₂ CH (CH ₂ OCH ₂ Ar) O), 69.19-68.67 (OCH ₂ CH ( C H ₂ O C H) ₂ Ar) O), 63.51 (OCH ₂ C H (CH ₂ O C H ₂ Ar) O) ppm.

The resulting polymer was characterized by size exclusion chromatography (SEC). SEC (DMF): M _n = 2.6 kDa, M _w = 2.7 kDa, Ð _M = 1.04.

The results of Example 25 are summarized in Table 15 below.

Table 15

P (NMMO-Co-MA)

Example 26

Poly (2 - [[(2-nitrophenyl) methoxy] methyl] oxirane-co-maleic anhydride) (poly (NMMO- nose -MA)) ⁷ Synthesis of

Poly (2-[[(2-nitrophenyl) methoxy] methyl] oxirane- co -maleic anhydride) (poly (NMMO- co- MA)) is synthesized as shown in Scheme 21 below.

Scheme 21

Poly (2-[[(2-nitrophenyl) methoxy] methyl]

Synthesis and isomerization of oxirane-co-maleic anhydride) (poly (NMMO-co-MA))

Ampoules were purified using standard Schlenk line technology using Mg (BHT) ₂ (THF) ₂ (121.4 mg, 0.2 mmol), propargyl alcohol (0.01 mL, 0.2 mmol), o -nitrobenzyl alcohol (2.48 mL, 0.5 mmol ) And maleic anhydride (490.3 mg, 0.5 mmol). The reagent was dissolved in toluene at a total monomer concentration of 2 M. The ampoule was sealed and heated to 80 ° C. for 24 hours. The obtained polymer was recovered by precipitation in excess diethyl ether. After centrifugation, the crude product was dissolved in chloroform, then diethylamine was added to the mixture, which was then refluxed for 24 h under N ₂ . Finally, the mixture was washed with excess phosphate buffered saline solution (0.5 M). The organic layers were combined and dried to yield poly (NMMO- co- MA) (Scheme 21).

The resulting polymer was characterized by: ¹ H NMR (300 MHz, 303 K, CDCl ₃ ): δ = 7.98-7.38 (m, Ar), 6.27 (m, CO H C = C H CO), 5.54 -5.34 (d, OCH ₂ C H (CH ₂ OCH ₂ ArNO ₂ ) O), 4.86 (s, OCH ₂ CH (CH ₂ OC H ₂ ArNO ₂ ) O), 4.41 (s, OC H ₂ CH (CH ₂ OCH ₂ ArNO ₂ ) O), 3.76 (m, OCH ₂ CH (C H ₂ OCH ₂ ArNO ₂ ) O) ppm. The resulting polymer was characterized by: ¹³ C NMR (125 MHz, 303K, CDCl ₃ ): δ = 163.99 (m, O- C = 0), 147.47 (m, Ar), 133.68 (m, -C = C- ), 129.19-124.76 (m, Ar), 71.93-71.22 (d, OCH ₂ CH (CH ₂ O C H ₂ Ar) O), 70.16 (s, O C H ₂ CH (CH ₂ OCH ₂ Ar ) O), 69.19-68.67 (d, OCH ₂ CH ( C H ₂ O C H ₂ Ar) O), 63.51 (d, OCH ₂ C H (CH ₂ O C H ₂ Ar) O) ppm.

In view of the foregoing, it should be understood that the present invention significantly advances the technology by providing functionalized poly (propylene fumarate) polymers that have been structurally and functionally improved in many ways. While specific embodiments of the invention have been disclosed in detail herein, it should be understood that the invention is not to be limited thereto or by modifications to the invention herein, as will be readily appreciated by those skilled in the art. . The scope of the invention will be understood by the following claims.

Claims (41)

Terminal or monomer functionalized poly (propylene fumarate) polymer. The terminal or monomeric functionalized poly (propylene fumarate) polymer of claim 1, comprising isomerized residues of the maleic anhydride monomer and residues of the functionalized propylene oxide monomer. The terminal or monomeric functionalized poly (propylene fumarate) polymer of claim 1, further comprising a moiety of the functionalized starting alcohol. The terminal or monomer functionalized poly (propylene fumarate) polymer of claim 1 having the formula:

Wherein n is an integer from 1 to 1000; R is a functional group or an alkyl or aryl group containing the functional group, wherein the functional group is a benzyl group, alkyn group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctin group, cyclooctyne group, ketone group , Aldehyde group, tertiary halogen group, and combinations thereof. The terminal or monomeric functionalized poly (propylene fumarate) polymer of claim 1, having the formula:

In formula, n is an integer of 1-1000. 6. The functionalized starting alcohol of claim 1, wherein the functionalized starting alcohol is propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one. 7. , 3-hydroxypropan-2-one, 5-hydroxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one Terminal or monomeric functionalized poly (propylene fumarate) polymer, selected from the group consisting of 5-norbornene-2-ol, α-bromoisobutyryl 4-methanol benzylmethanoate, and combinations thereof. 4. The functionalized propylene oxide monomer of claim 2, wherein the functionalized propylene oxide monomer is alkyne functionalized propylene oxide, 0- benzyl functionalized propylene oxide, 2-[[(2-nitrophenyl) methoxy] methyl] oxy A terminal or monomeric functionalized poly (propylene fumarate) polymer selected from the group consisting of lan (NMMO), glycidyl propargyl ether, (±) -epichlorohydrin, and combinations thereof. The functionalized starting alcohol according to any one of claims 2 to 6, wherein the functionalized starting alcohol is an alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde. A terminal or monomeric functionalized poly (propylene fumarate) polymer comprising a functional group selected from the group consisting of groups, tertiary halogen groups, and combinations thereof. The terminal or monomer functionalized poly (propylene fumarate) polymer according to any one of claims 1 to 3 and 6 to 8 comprising both terminal functional groups and monomer functional groups. The terminal or monomeric functionalized poly (propylene fumarate) polymer according to any one of claims 1 to 3 and having the formula:

Wherein n is an integer from 1 to 100; R is a first functional group or an alkyl or aryl group containing the first functional group, wherein the first functional group is an alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctin group, cyclooctyne group , Ketone group, aldehyde group, tertiary halogen group, and poly (ethylene glycol) group, and combinations thereof; R 'is a second functional group or an alkyl or aryl group containing said second functional group, said second functional group being an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, a thiol group, a halide group, and these Is selected from the group consisting of The terminal or monomeric functionalized poly (propylene fumarate) polymer according to any one of claims 1 to 3 and having a formula selected from:

Wherein n is an integer from about 1 to about 100. The terminal or monomeric functionalized poly (propylene fumarate) polymer of claim 1 having a number average molecular weight of about 0.7 kDa to about 100,000 kDa. The terminal or monomeric functionalized poly (propylene fumarate) polymer of claim 1 having a polydispersity index ( Ð _M ) of about 1.01 to about 1.8. Claims 1 to 3, 6 to 12, and including glycidyl groups linked via ether linkages to alkyl or aryl groups comprising functional groups which can be introduced into the click reaction with corresponding functional groups, and Functionalized propylene oxide monomer for forming the monomer functionalized poly (propylene fumarate) polymer as claimed in claim 13. 15. The group of claim 14, wherein said alkyl or aryl group comprising a functional group capable of introducing a click reaction together with a corresponding functional group consists of an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, or a combination thereof. Functionalized monomers selected from. The functionalized monomer of claim 14 or 15, wherein the functionalized monomer is glycidyl propargyl ether. The functionalized monomer of claim 14, wherein the functionalized monomer has the formula

. 16. The functionalized monomer of claim 14 or 15, wherein said functionalized monomer is 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO). 19. The functionalized monomer of any one of claims 14, 15 and 18 having the formula:

. The functionalized monomer of claim 14 having the formula:

Wherein R is a functional group or an alkyl or aryl group comprising said functional group, wherein said functional group is from an alkyne group, an alkene group, a hydroxyl group, a protected hydroxyl group, a thiol group, a halide group, and combinations thereof Is selected. A 3-D printed polymer scaffold comprising a functionalized poly (propylene fumarate) polymer as claimed in claim 1. The 3-D printed polymer scaffold of claim 21, further comprising a plurality of bioactive materials or functional moieties bound to the functionalized poly (propylene fumarate) polymer. 14. A process for the preparation of terminal or monomeric functionalized poly (propylene fumarate) polymers as claimed in any of claims 1 to 13.
A. Preparing a starting alcohol, wherein the starting alcohol further comprises a functional end group;
B. combining the starting alcohol, magnesium catalyst, maleic anhydride, and propylene oxide in a suitable vessel and adding a suitable solvent;
C. Sealing the vessel and then heating to cause and / or maintain a ring-opening polymerization reaction between maleic anhydride and propylene oxide initiated by the starting alcohol, thereby containing the poly (propylene male) Ate) forming a polymer;
D. collecting and purifying the poly (propylene maleate) polymer comprising said functional end groups; And
E. isomerizing the poly (propylene maleate) polymer comprising the functional end group with respect to the poly (propylene fumarate) polymer comprising the functional end group. The method of claim 23, wherein the molar ratio of the starting alcohol to the magnesium catalyst in the combination of Step B is about 1: 1. The method of claim 23, wherein the molar ratio of the starting alcohol to the total moles of maleic anhydride and propylene oxide in the combination of Step B is from about 1: 5 to about 1: 1000. The method of claim 23, wherein the total concentration of maleic anhydride and propylene oxide in the solution of Step B is from about 0.5M to about 5.0M. The method of claim 23, wherein the suitable solvent is toluene or hexane. The method of claim 23, wherein the heating step comprises heating the vessel to a temperature of about 40 ° C. to about 100 ° C. 24. The method of claim 23, wherein the suitable solvent is hexane and the heating step comprises heating the vessel to a temperature of about 45 ° C. 25. The method of claim 23, wherein the starting alcohol is benzyl alcohol, propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropan-2-one, 5 -Hydroxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, α- Bromoisobutyryl 4-methanol benzylmethanoate, and combinations thereof. The functional terminal group of claim 23, wherein the functional terminal group of the starting alcohol is benzyl group, alkyne group, propargyl group, allyl group, alkene group, 4-dibenzocyclooctyne group, cyclooctyne group, ketone group, aldehyde group, 3 Primary halogen groups, poly (ethylene glycol) groups, and combinations thereof. The method of claim 23, wherein the magnesium catalyst comprises Mg (BHT) ₂ (THF) ₂ . 14. A process for the production of a functionalized poly (propylene fumarate) polymer as claimed in any one of claims 1 to 3, 6 to 12 and 13.
A. preparing functionalized propylene oxide;
B. reacting the functionalized propylene oxide with maleic anhydride and starting alcohol in the presence of a magnesium catalyst to form a functionalized poly (propylene maleate) polymer;
C. isomerizing the polymer by reacting the functionalized poly (propylene maleate) polymer with a base to form the functionalized poly (propylene fumarate) polymer of claim 1. 34. The composition of claim 33, wherein the functionalized propylene oxide is alkyne functionalized propylene oxide, 0- benzyl functionalized propylene oxide, 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO), Glycidyl propargyl ether, (±) -epichlorohydrin and combinations thereof. The method of claim 33, wherein the starting alcohol is benzyl alcohol, propargyl alcohol, allyl alcohol, 4-dibenzocyclooctinol, 4-hydroxybutan-2-one, 3-hydroxypropan-2-one, 5 -Hydroxypentan-2-one, 6-hydroxyhexan-2-one, 7-hydroxyheptan-2-one, 8-hydroxyoctan-2-one, 5-norbornene-2-ol, α- And a functionalized starting alcohol selected from bromoisobutyryl 4-methanol benzylmethanoate, and combinations thereof. The method of claim 33, wherein the metal catalyst is magnesium 2,6- di -tert-butyl-4-methylphenoxide (Mg (BHT) ₂ (THF) ₂ ). A process for preparing the functionalized monomer of claim 16, wherein
A. adding propargyl alcohol to an aqueous solution containing a base selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), and combinations thereof;
B. dissolving (±) -epichlorohydrin and tetrabutylammonium hydrogensulfate in a suitable organic solvent;
C. adding the solution of step B and H ₂ O to the propargyl alcohol solution of step A; And
D. conducting the reaction under an inert atmosphere to produce glycidyl propargyl ether. 38. The method of claim 37, wherein the step of adding propargyl alcohol (step A) comprises adding dropwise propargyl alcohol to an aqueous solution of sodium hydroxide (NaOH) at a temperature of about -10 ° C to about 30 ° C with stirring, The aqueous solution of sodium hydroxide (NaOH) comprises about 20% to about 50% by weight of NaOH;
The organic solvent in the dissolution step (step B) comprises hexane; And
Advancing the reaction (step D) comprises increasing the reaction temperature to ambient temperature and continuing the reaction for about 1 hour to about 24 hours under an N ₂ blanket. The method of claim 37 or 38,
E. quenching the reaction;
F. extracting the crude product with a suitable organic solvent; And
G. Purifying the crude product of step F by column chromatography or distillation to produce purified glycidyl propargyl ether. 20. A process for the preparation of functionalized monomers as claimed in claim 18 or 19,
A. dissolving o -nitrobenzyl alcohol in a suitable organic solvent;
B. adding an aqueous solution containing tetrabutylammonium hydrogensulfate and a base to the o -nitrobenzyl alcohol solution of step A, the base comprising sodium hydroxide (NaOH), potassium hydroxide (KOH), and combinations thereof Selected from the group consisting of;
C. adding (±) -epichlorohydrin; And
D. progressing the reaction to produce 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO). The method of claim 40,
E) extracting the crude product with a suitable organic solvent; And
F) purifying the crude product of step D by column chromatography or distillation to produce purified 2-[[(2-nitrophenyl) methoxy] methyl] oxirane (NMMO).

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